Anti-obesity agents

ABSTRACT

The present invention relates to a compound comprising a PYY peptide or a functional derivative thereof, which is coupled to a reactive group. Such a reactive group is capable of reacting on a blood component so as to form a stable covalent bond therewith. The present invention also relates to a conjugate comprising such a compound which is covalently bonded to a blood component. Moreover, the invention also relates to a method of enhancing, in a patient, the anti-obesity activity of a PYY peptide or functional derivative thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation-in-Part of U.S. patentapplication Ser. No. 09/657,276 filed on Sep. 7, 2000, which claimspriority to U.S. Provisional Patent Application No. 60/153,406 filed onSep. 10, 1999, and to U.S. Provisional Patent Application No. 60/159,783filed on Oct. 15, 1999. This application is also a Continuation-in-PartApplication of U.S. patent application Ser. No. 11/040,810 filed Jan.21, 2005, which is a Continuation Application of U.S. patent applicationSer. No. 10/471,348 filed on Sep. 8, 2003, which is a National Stage ofInternational Patent Application No. PCT/CA03/01097 filed on Jul. 29,2003, which claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application Ser. No. 60/400,199 filed on Jul. 31,2002, and U.S. Provisional Patent Application Ser. No. 60/400,413 filedon Jul. 31, 2002. U.S. patent application Ser. No. 11/040,810 is also aContinuation-in-Part Application of U.S. patent application Ser. No.09/623,548 filed on May 17, 2000, now U.S. Pat. No. 6,849,714, which wasa National Stage of International Application No. PCT/US00/13576, filedon May 17, 2000, which claims priority to U.S. Provisional PatentApplication No. 60/134,406 filed on May 17, 1999, U.S. ProvisionalPatent Application No. 60/153,406 filed on Sep. 10, 1999, and to U.S.Provisional Patent Application No. 60/159,783 filed on Oct. 15, 1999.The above-mentioned applications are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to compounds and methods for treatingeating disorders or metabolic syndromes. More particularly, the presentrelates to peptides, conjugates and methods for treating obesity.

BACKGROUND OF THE INVENTION

A number of postprandial endocrine, paracrine or autocrine messengerproducts involved in the signaling of hunger and satiety that arepresent in circulation such as hormones or peptides. The results from anelevated or reduced plasma concentration of one or more of theseproducts will have either global orexigenic or anorexigenic effects.

Examples of peptides associated with anorexigenic effects are pancreaticpolyeptide (PP), neuropeptide Y (NPY) and peptide YY (PYY).

These peptides act through Y receptors for which five are known, Y1, Y2,Y3, Y4 and Y5 and regulate pancreatic secretion, gastric emptying andgastric motility. The Y receptors are found throughout the peripheraland central nervous systems as well as on various gastrointestinal organcells.

Pancreatic polypeptide is secreted in the pancreas and helps controlenergy homeostasis through inhibition of pancreatic secretions such asfor example insulin thus leading to an increased blood glucose level andsignaling a need for reduced feeding.

Hypothalamic secreted neuropeptide Y participates in the control of foodintake through binding and activation Y1 and possibly Y2 and Y5receptors.

One of the most discussed examples in recent times is PYY₁₋₃₆. It isproduced in endocrine L cells lining the distal small bowel and colon.The prepro PYY is clipped by signal peptidases to give proPYY₁₋₇₀. Thispeptide is further modified by prohormone dibasic convertase leading toPYY-Gly-Lys-Arg followed by Carboxypeptidase B to give PYY-Gly andfinally to PYY₁₋₃₆ by amidation enzyme. It is then released from thecell where a metabolic derivative obtained through DPP-IV cleavage ofthe two N-terminal amino acids give circulating PYY₃₋₃₆.

PYY₁₋₃₆ binds and activates Y1, Y2 and Y5 receptors found on a varietyof cells surfaces as for NPY. The cells are found peripherally in thegastrointestinal tract as well as on the arcuate nucleus. The result ofinteraction with the Y2 found on the arcuate is thought to lead to acentral nervous system response. Alternatively the Y2 receptors foundperipherally on the surface of cell within the gastrointestinal tracthave been shown to have an effect on gastric motility, gastric acidsecretion and intestinal motility. The result of these interactions leadto reduced food and caloric intake.

Unlike PYY₁₋₃₆ which interacts equally with the Y1 and Y2 receptors,PYY₃₋₃₆ is selective to the Y2 receptor. A selective agonist of the Y2receptor has been demonstrated to be beneficial as compared to a broadagonist. In fact, the Y1 receptor has been associated with hypertension(A. Balasubramaniam et al. J. Med. Chem. 2000, 43, 3420-27,Balasubramaniam A et al. Pept Res. 1988 1, 32-5). PYY₃₋₃₆ has beendemonstrated to reduce food intake in vivo (Nature, 2002, 418, 650-4).

The advantage of using PYY₃₋₃₆ is that it is a natural appetitecontrolling hormone. There will not psychological side effect from thecentral nervous system such as when norepinephrine and serotoninreuptake inhibitor or other stimulants are used. Another advantage isthat this class of therapeutic agent does not interfere with theabsorption of certain nutritional or fat containing elements such asgastrointestinal lipase inhibitor that cause uncomfortable side effects.An inconvenience of using PYY₃₋₃₆ is need for multiple dailyadministrations.

A new anti-obesity agent that has an enhanced activity and which wouldpermit to avoid the above-mentioned drawbacks would therefore be highlydesired. A method for enhancing the anti-obesity activity of a PYYpeptide or a functional derivative thereof would also be desired.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided a compoundcomprising a PYY peptide or a functional derivative thereof which iscoupled to a reactive group, the reactive group being capable ofreacting with an amino group, a hydroxyl group or a thiol group on ablood component so as to form a stable covalent bond therewith, therebysubstantially preventing the PYY peptide or functional derivativethereof from crossing the blood brain barrier.

According to another aspect of the invention, there is provided aconjugate comprising

-   -   a blood component; and    -   a PYY peptide or a functional derivative thereof which is        coupled to a reactive group,        wherein the reactive group is coupled with at least an amino        group, a hydroxyl group or a thiol group on the blood component        so as to form a stable covalent bond therewith, thereby        substantially preventing the PYY peptide or derivative thereof        from crossing the blood brain barrier.

It should be understood that, in the compounds and conjugates of thepresent invention, the stable covalent bond between the PYY peptide orfunctional derivative thereof and the blood component can be formed invivo or ex vivo.

According to another aspect of the invention, there is provided a methodof enhancing, in a patient, the anti-obesity activity of a PYY peptideor functional derivative thereof comprising the step of covalentlybonding the PYY peptide or functional derivative thereof to a bloodcomponent, thereby preventing the PYY peptide or functional derivativethereof from crossing the blood brain barrier when administered to thepatient, wherein preventing the PYY peptide or functional derivativethereof from crossing the blood brain barrier results in an enhancedanti-obesity activity of the PYY peptide or functional derivativethereof.

According to another aspect of the invention, there is provided in amethod for treating obesity by administering a PYY peptide or afunctional derivative thereof to a patient, the improvement wherein thePYY peptide or functional derivative thereof is covalently bonded to ablood component so as to prevent the PYY peptide or functionalderivative thereof from crossing the blood brain barrier, therebyenhancing its anti-obesity activity.

It should also be understood that, in the methods of the presentinvention, the covalent bonding between the PYY peptide or thefunctional derivative thereof and the blood component can be formed invivo or ex vivo.

According to another aspect of the invention, there is provided acompound comprising a peptide of formula:X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁- (SEQ ID NO: 1)X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉- X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-Awherein

-   -   X₁ is absent, tyr or ala;    -   X₂ is absent or pro;    -   X₃ is absent, lys or an analog thereof, ile, leu, or ala;    -   X₄ is absent, lys or an analog thereof, or glu;    -   X₅ is absent or pro;    -   X₆ is absent, glu, val or asp;    -   X₇ is absent, ala, tyr or asn;    -   X₈ is absent or pro;    -   X₉ is absent or gly;    -   X₁₀ is absent, glu or asp;    -   X₁₁ is absent, asp or asn;    -   X₁₂ is absent, lys or an analog thereof, or ala;    -   X₁₃ is absent, lys or an analog thereof, ser, thr, or pro;    -   X₁₄ is absent, lys or an analog thereof, ala, or pro;    -   X₁₅ is absent, lys or an analog thereof, or glu;    -   X₁₆ is absent, glu, gln or asp;    -   X₁₇ is absent, leu or met;    -   X₁₈ is absent, lys or an analog thereof, ser, or ala;    -   X₁₉ is absent, arg or gln;    -   X₂₀ is absent or tyr;    -   X₂₁ is absent, tyr or ala;    -   X₂₂ is ala or ser;    -   X₂₃ is ser, asp or ala;    -   X₂₄ is leu;    -   X₂₅ is arg or lys;    -   X₂₆ is his, arg or lys;    -   X₂₇ is tyr;    -   X₂₈ is leu or ile;    -   X₂₉ is asn    -   X₃₀ is leu or met;    -   X₃₁ is val, leu or ile;    -   X₃₂ is thr;    -   X₃₃ is arg or lys;    -   X₃₄ is gln or pro;    -   X₃₅ is arg or lys;    -   X₃₆ is tyr or a derivative thereof; and    -   A is absent lys or a derivative thereof, and        at least one reactive group coupled to any one of X₁ to X₃₆ and        A, directly or via a linking group.

According to another aspect of the invention, there is provided aconjugate comprising a blood component and a compound having a peptideof formula: X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁- (SEQ ID NO: 1)X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉- X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-Awherein X₁-X₃₆ and A are as previously defined, and a reactive groupcoupled to any one of X₁-X₃₆ and A, directly or via a linking group, andwherein the reactive group is coupled with at least an amino group, ahydroxyl group or a thiol group on the blood component so as to form astable covalent bond therewith.

It has been found that the compounds and conjugates of the presentinvention demonstrated an enhanced anti-obesity activity with respect toPYY peptides such as PYY₁₋₃₆ and PYY₃₋₃₆. It also has been found thatthese compounds and conjugates are efficient for reducing the foodconsumption of a subject, thereby treating or preventing obesity.

It has been found that the methods of the present invention areeffective for enhancing the anti-obesity activity of PYY peptide or aderivative thereof and/or for treating obesity. It also has been foundthat by preventing the compounds or conjugates from crossing the bloodbrain barrier, an enhanced anti-obesity activity of the PYY peptides orderivative thereof was observed.

The expression “a PYY peptide or a functional derivative thereof” asused herein refers to a PYY peptide such as PYY₁₋₃₆ or PYY₃₋₃₆ or to afunctional derivative of the PYY peptide. Such a functional derivativewould be understood by a person skilled in the art as a derivative whichsubstantially maintains the activity of the PYY peptide. Preferably,such a functional derivative has an in vitro NPY Y2 receptor bindingactivity which is at least 1/100 of the in vitro NPY Y2 receptor bindingactivity of PYY₃₋₃₆. More preferably, the functional derivative has anin vitro NPY Y2 receptor binding activity which is equal or superior tothe in vitro NPY Y2 receptor binding activity of PYY₃₋₃₆. In anon-limitative manner, the functional derivative can comprise a peptideof the following formula:X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-A(SEQ ID NO: 1) or Z₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀-Z₁₁-Z₁₂ (SEQ ID NO: 2)wherein X₁ to X₃₆, and A are as previously defined, and wherein Z₁ isala, Z₄ is arg, Z₈ is asn, Z₁₂ is arg, and Z₂, Z₃, Z₅ to Z₇ and Z₉ toZ₁₁ are selected from the group consisting of the natural amino acids.

The expression “lys or an analog thereof” refers to a lysine or ananalog thereof that will substantially maintains the activity of thepeptide. In a non-limitative manner, the lys analog can be of formula:

where n is an integer having a value of 0, 1, 2, 3 or 4.

The expression “tyr or a derivative thereof” refers to a tyrosine or aderivative thereof that will substantially maintains the activity of thepeptide. In a non-limitative manner, the tyr derivative can be offormula:

where

-   R₁ is H, a protecting group (PG), a C₁-C₁₀ branched, linear or    cyclic alkyl, a phosphate or a sulfate; R₂ and R₃ are same or    different and selected from the group consisting of H, D and I; and    R₄ is OH, OPG, OR₅, SH, SPG, SR₅, NH₂, NHPG, N(PG)₂, N(R₅)₂, NR₅PG,    or NHR₆, where R₅ is a C₁-C₁₀ branched, linear or cyclic alkyl, and    R₆ is a solid phase support.

The expression “protecting group (PG)” as used herein refers to suitableprotecting groups as defined in T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 3rd Edition, (1999) John Wiley &Sons, which is hereby incorporated by reference. The person skilled inthe art will understand that nature of the protecting group will varyaccording to the functionality that has to be protected. Greene et al.discloses, as example, various protecting groups for carboxylic acids,alcohols, thiols, amines, amides etc.

The expression “lys or a derivative thereof” refers to a lysine or aderivative thereof that will substantially maintains the activity of thepeptide. In a non-limitative manner, the lys derivative can be offormula:

where

-   R₄ is OH, OPG, OR₅, SH, SPG, SR₅, NH₂, NHPG, N(PG)₂, N(R₅)₂, NR₅PG,    or NHR₆, where R₅ is a C₁-C₁₀ branched, linear or cyclic alkyl, and    R₆ is a solid phase support; and n is an integer having a value of    0, 1, 2, 3 or 4.

The PYY peptide or functional derivative thereof can be selected fromSEQ IDS NO: 1 to 15, preferably from SEQ IDS NO: 2 to 13, and morepreferably from SEQ ID NO: 4.

In the compounds and conjugates of present invention, there ispreferably only one reactive group. Advantageously, the reactive groupis coupled to any one of X₁ to X₂₁, X₂₃, X₂₄, X₂₆ to X₂₈, X₃₀ to X₃₂,X₃₄ to X₃₆, and A. Alternatively, the reactive group can be coupled toany one of Z₁ to Z₁₂ and preferably to any one of Z₂, Z₃, Z₅ to Z₇ andZ₉ to Z₁₁. The reactive group can also be connected to the peptide orthe PYY functional derivative by means of a linking group.

According to preferred embodiments, in the compounds and conjugates ofthe invention which comprise a peptide of formula:X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-A(SEQ ID NO: 1):

-   X₁ can be absent, the reactive group, linking group-(reactive    group), tyr or ala, the tyr or ala being optionally coupled to the    reactive group or to the linking group-(reactive group). Preferably,    X₁ is absent. X₂ can be absent, pro, the reactive group or the    linking group-(reactive group). Preferably, X₂ is absent. X₃ can be    absent, lys or an analog thereof, (reactive group)-lys, (reactive    group)-linking group-lys, (reactive group)-lys analog, (reactive    group)-linking group-lys analog, ile, leu, or ala, wherein the    reactive group is coupled to the free amine of lys or lys analog.    Preferably, X₃ is leu. X₄ can be absent, lys or an analog thereof,    (reactive group)-lys, (reactive group)-linking group-lys, (reactive    group)-lys analog, (reactive group)-linking group-lys analog, or    glu, wherein the reactive group is coupled to the free amine of lys    or lys analog. Preferably, X₄ is lys (reactive group)-lys or    (reactive group)-linking group-lys. X₅ is preferably pro. X₆ is    preferably glu. X₇ is preferably ala. X₈ is preferably pro. X₉ is    preferably gly. X₁₀ is preferably glu. X₁₁ is preferably asp. X₁₂    can be absent, lys or an analog thereof, (reactive group)-lys,    (reactive group)-linking group-lys, (reactive group)-lys analog,    (reactive group)-linking group-lys analog, or ala, wherein the    reactive group is coupled to the free amine of lys or lys analog.    Preferably, X₁₂ is ala. X₁₃ can be absent, lys or an analog thereof,    (reactive group)-lys, (reactive group)-linking group-lys, (reactive    group)-lys analog, (reactive group)-linking group-lys analog, ser,    thr, or pro, wherein the reactive group is coupled to the free amine    of lys or lys analog. Preferably, X₁₃ is ser. X₁₄ can be absent, lys    or an analog thereof, (reactive group)-lys, (reactive group)-linking    group-lys, (reactive group)-lys analog, (reactive group)-linking    group-lys analog, ala or pro, wherein the reactive group is coupled    to the free amine of lys or lys analog. X₁₄ is preferably pro. X₁₅    can be absent, lys or an analog thereof, (reactive group)-lys,    (reactive group)-linking group-lys, (reactive group)-lys analog,    (reactive group)-linking group-lys analog, or glu, wherein the    reactive group is coupled to the free amine of lys or lys analog.    X₁₅ is preferably glu. X₁₆ is preferably glu. X₁₇ is preferably leu.    X₁₈ can be absent, lys or an analog thereof, (reactive group)-lys,    (reactive group)-linking group-lys, (reactive group)-lys analog,    (reactive group)-linking group-lys analog, ser, or ala, wherein the    reactive group is coupled to the free amine of lys or lys analog.    X₁₈ is preferably ser. X₁₉ is preferably arg. X₂₀ is preferably tyr.    X₂₁ can be absent, tyr, ala, a reactive group, or linking    group-(reactive group), wherein the linking group is coupled to X₂₀    and X₂₂. X₂₁ is preferably tyr. X₂₂ is preferably ala. X₂₃ is    preferably ser. X₂₅ is preferably arg. X₂₆ is preferably his. X₂₈ is    preferably leu. X₃₀ is preferably leu. X₃₁ is preferably val. X₃₃ is    preferably arg. X₃₄ is preferably gln. X₃₅ is preferably arg.

In a preferred embodiment of the present invention, the reactive groupcan be selected from the group consisting of Michael acceptors(preferably an unsaturated carbonyl such as a vinyl carbonyl or a vinylsulfone moiety), succinimidyl-containing groups (such as,N-hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS)etc.), an electrophilic thiol acceptor (such as pyridyldithio (Pyr-S—S),an alpha halogenated alkyl carbonyl (such as an alpha halogenated alkylcarbonyl where the alkyl, further to the halogen substituent, maycontains or not a substituent such as a C₁-C₈ alkyl or phenyl), andmaleimido-containing groups (such as gamma-maleimide-butyralamide(GMBA), beta-maleimidopropionic acid (MPA), alpha-maleimidoacetic acid(MAA) etc.). Advantageously, the reactive group is amaleimido-containing group. Alternatively, the reactive group isadvantageously an alpha halogenated alkyl carbonyl and preferably alphaiodo acetyl. Preferably, the reactive group is a reactive group, whichis capable of reacting with an amino group, a hydroxyl group or a thiolgroup on a blood component so as to form a stable covalent bond.

As example, the maleimido group is most selective for sulfhydryl groupson peptides when the pH of the reaction mixture is kept between 6.5 and7.4. At pH 7.0, the rate of reaction of maleimido groups withsulfhydryls is 1000-fold faster than with amines. A stable thioetherlinkage between the maleimido group and the sulfhydryl is formed whichcannot be cleaved under physiological conditions. Primary amines can bethe principal targets for NHS esters. Accessible α-amine groups presenton the N-termini of proteins can react with NHS esters. However, α-aminogroups on a protein may not be desirable or available for the NHScoupling. While five amino acids have nitrogen in their side chains,only the ε-amine of lysine reacts significantly with NHS esters. Anamide bond can be formed when the NHS ester conjugation reaction reactswith primary amines releasing N-hydroxysuccinimide.

In a preferred embodiment of the present invention, the reactive groupis coupled to an amino acid of the peptide via a linking group (orlinker), such as, but not limited to (2-amino) ethoxy acetic acid (AEA),ethylenediamine (EDA), amino ethoxy ethoxy succinimic acid (AEES),AEES-AEES, 2-[2-(2-amino)ethoxy)] ethoxy acetic acid (AEEA), AEEA-AEEA,—NH₂—(CH₂)_(n)-COOH where n is an integer between 1 and 20 and alkylchain (C₁-C₁₀) motif saturated or unsaturated in which could beincorporated oxygen nitrogen or sulfur atoms, such as, but not limitedto glycine, 3-aminopropionic acid (APA), 8-aminooctanoic acid (OA) and4-aminobenzoic acid (APhA) and combinations thereof.

In a preferred embodiment of the present invention, the blood componentis a blood protein, more preferably is albumin (such as human serumalbumin (HSA)).

Preferably, the invention relates to anti-obesity agents such as PYY₃₋₃₆or derivatives thereof, which can be shortened versions of the latter.The new bioconjugates formed by the ex vivo, in vivo or in vitrocovalent bonding between the peptides of the present invention and ablood component have been found to be very selective to the neuropeptideY2 receptor.

PP and NPY peptides could also be suitable as an alternative to PYY orits functional derivatives in the various embodiments of the presentinvention.

The methods of the present invention include extending the effectivetherapeutic life of the conjugated anti-obesity peptide derivatives ascompared to administration of the unconjugated peptide to a patient.Moreover, the anti-obesity activity of the conjugated anti-obesitypeptide derivatives of the present invention is considerably enhanced ascompared the unconjugated peptide to a patient peptides of the presentinvention. The derivatives or modified peptides can be of a typedesignated as a DAC™ (Drug Affinity Complex), which comprises theanti-obesity peptide molecule and a linking group together with achemically reactive group capable of reaction with a reactivefunctionality of a mobile blood protein. By reaction with the bloodcomponent or protein the modified peptide, or DAC, may be delivered viathe blood to appropriate sites or receptors. Moreover, conjugating thepeptides to a blood component provides a protection against thedegradation of enzymes.

A. Specific Labeling.

Preferably, the compounds, derivatives or modified peptides of thisinvention are designed to specifically react with thiol groups on mobileblood proteins. Such a reaction is preferably established by covalentbonding of the peptide modified with a maleimido-containing group linkedto a thiol group on a mobile blood protein such as serum albumin or IgG.

Under certain circumstances, specific labeling with maleimido-containinggroup offers several advantages over non-specific labeling of mobileproteins with groups such as NHS and sulfo-NHS. Thiol groups are lessabundant in vivo than amino groups. Therefore, the compounds of thepresent invention such as maleimido-modified peptides, can covalentlybond to fewer proteins. For example, in albumin (an abundant bloodprotein) there is only a single thiol group. Thus,peptide-(maleimido-containing group)-albumin conjugates can tend tocomprise a 1:1 molar ratio of peptide to albumin. In addition toalbumin, IgG molecules (class II) also have free thiols. Since IgGmolecules and serum albumin make up the majority of the soluble proteinin blood they also make up the majority of the free thiol groups inblood that are available to covalently bond to maleimide-modifiedpeptides.

Further, even among free thiol-containing blood proteins, includingIgGs, specific labeling with a maleimido-containing group leads to thepreferential formation of peptide-(maleimido-containing group)-albuminconjugates, due to the unique characteristics of albumin itself. Thesingle free thiol group of albumin, highly conserved among species, islocated at amino acid residue 34 (Cys34). It has been demonstratedrecently that the Cys34 of albumin has increased reactivity relative tofree thiols on other free thiol-containing proteins. This is due in partto the very low pK value of 5.5 for the Cys34 of albumin. This is muchlower than typical pK values for cysteine residues in general, which aretypically about 8. Due to this low pK, under normal physiologicalconditions Cys34 of albumin is predominantly in the anionic form, whichdramatically increases its reactivity. In addition to the low pK valueof Cys34, another factor, which enhances the reactivity of Cys34 is itslocation in a crevice close to the surface of one loop of region V ofalbumin. This location makes Cys34 very available to ligands of allkinds, and is an important factor in Cys34's biological role as a freeradical trap and a free thiol scavenger. These properties make Cys34highly reactive toward maleimide-peptides, and the reaction rateacceleration can be as much as 1000-fold relative to rates of reactionof maleimide-peptides with other free-thiol containing proteins.

Another advantage of peptide-(maleimido-containing group)-albuminconjugates is the reproducibility associated with the 1:1 loading ofpeptide to albumin specifically at Cys34. Other techniques, such asglutaraldehyde, DCC, EDC and other chemical activations of, e.g, freeamines, lack this selectivity. For example, albumin contains 52 lysineresidues, 25 to 30 of which are located on the surface of albumin andtherefore accessible for conjugation. Activating these lysine residues,or alternatively modifying peptides to couple through these lysineresidues, results in a heterogenous population of conjugates. Even ifstatistical 1:1 molar ratios of peptide to albumin are employed, theyield will consist of multiple conjugation products, some containing 0,1, 2 or more peptides per albumin, and each having peptides randomlycoupled at any one or more of the 25 to 30 available lysine sites. Giventhe numerous possible combinations, characterization of the exactcomposition and nature of each conjugate batch becomes difficult, andbatch-to-batch reproducibility is all but impossible, making suchconjugates less desirable as a therapeutic. Additionally, while it wouldseem that conjugation through lysine residues of albumin would at leasthave the advantage of delivering more therapeutic agent per albuminmolecule, studies have shown that a 1:1 ratio of therapeutic agent toalbumin is preferred. In an article by Stehle, et al., “The Loading RateDetermines Tumor Targeting properties of Methotrexate-Albumin Conjugatesin Rats,” Anti-Cancer Drugs, Vol. 8, pp. 677-685 (1988), the authorsreport that a 1:1 ratio of the anti-cancer methotrexate to albuminconjugated via amide coupling of one of the available carboxylic acidson methotrexate to any lysine on albumin gave the most promisingresults. The conjugates describe therein were preferentially taken up bytumor cells, whereas the conjugates bearing 5:1 to 20:1 methotrexatemolecules to albumin had altered HPLC profiles and were quickly taken upby the liver in vivo. It is postulated that at these higher ratios,confer conformational changes to albumin diminishing its effectivenessas a therapeutic carrier.

Through controlled administration of maleimido-peptides in vivo, one cancontrol the specific labeling of albumin and IgG in vivo. In typicaladministrations, 80-90% of the administered maleimido-peptides willlabel albumin and less than 5% will label IgG. Trace labeling of freethiols such as glutathione, cysteine or Cys-Gly will also occur. Suchspecific labeling is preferred for in vivo use as it permits an accuratecalculation of the estimated half-life of the administered agent.

In addition to providing controlled specific in vivo labeling,maleimide-peptides can provide specific labeling of serum albumin andIgG ex vivo. Such ex vivo labeling involves the addition ofmaleimide-peptides to blood, serum or saline solution containing serumalbumin and/or IgG. Once conjugation has occurred ex vivo with themaleimido-peptides, the blood, serum or saline solution can bereadministered to the patient's blood for in vivo treatment.

In contrast to NHS-peptides, maleimido-peptides are generally quitestable in the presence of aqueous solutions and in the presence of freeamines. Since maleimido-peptides will only react with free thiols,protective groups are generally not necessary to prevent themaleimido-peptides from reacting with itself. In addition, the increasedstability of the modified peptide permits the use of furtherpurification steps such as HPLC to prepare highly purified productssuitable for in vivo use. Lastly, the increased chemical stabilityprovides a product with a longer shelf life.

B. Non-Specific Labeling.

The anti-obesity peptides of the invention may also be modified fornon-specific labeling of blood components. Bonds to amino groups willalso be employed, particularly with the formation of amide bonds fornon-specific labeling. To form such bonds, one may use as a chemicallyreactive group a wide variety of active carboxyl groups, particularlyesters, where the hydroxyl moiety is physiologically acceptable at thelevels required. While a number of different hydroxyl groups may beemployed in these linking agents, the most convenient would beN-hydroxysuccinimide (NHS) and N-hydroxy-sulfosuccinimide (sulfo-NHS).

Other linking agents that may be utilized are described in U.S. Pat. No.5,612,034, which is hereby incorporated by reference. The various siteswith which the chemically reactive group of the modified peptides mayreact in vivo include cells, particularly red blood cells (erythrocytes)and platelets, and proteins, such as immunoglobulins, including IgG andIgM, serum albumin, ferritin, steroid binding proteins, transferrin,thyroxin binding protein, α-2-macroglobulin, and the like. Thosereceptors with which the modified peptides react, which are notlong-lived, will generally be eliminated from the human host withinabout three days. The proteins indicated above (including the proteinsof the cells) will remain at least three days, and may remain five daysor more (usually not exceeding 60 days, more usually not exceeding 30days) particularly as to the half life, based on the concentration inthe blood.

For the most part, reaction can be with mobile components in the blood,particularly blood proteins and cells, more particularly blood proteinsand erythrocytes. By “mobile” is intended that the component does nothave a fixed situs for any extended period of time, generally notexceeding 5 minutes, more usually one minute, although some of the bloodcomponent may be relatively stationary for extended periods of time.

Initially, there will be a relatively heterogeneous population offunctionalized proteins and cells. However, for the most part, thepopulation within a few days will vary substantially from the initialpopulation, depending upon the half-life of the functionalized proteinsin the blood stream. Therefore, usually within about three days or more,IgG will become the predominant functionalized protein in the bloodstream.

Usually, by day 5 post-administration, IgG, serum albumin anderythrocytes will be at least about 60 mole %, usually at least about 75mole %, of the conjugated components in blood, with IgG, IgM (to asubstantially lesser extent) and serum albumin being at least about 50mole %, usually at least about 75 mole %, more usually at least about 80mole %, of the non-cellular conjugated components.

The desired conjugates of non-specific modified peptides to bloodcomponents may be prepared in vivo by administration of the modifiedpeptides to the patient, which may be a human or other mammal. Theadministration may be done in the form of a bolus or introduced slowlyover time by infusion using metered flow or the like.

If desired, the subject conjugates may also be prepared ex vivo bycombining blood with modified peptides of the present invention,allowing covalent bonding of the modified peptides to reactivefunctionalities on blood components and then returning or administeringthe conjugated blood to the host. Moreover, the above may also beaccomplished by first purifying an individual blood component or limitednumber of components, such as red blood cells, immunoglobulins, serumalbumin, or the like, and combining the component or components ex vivowith the chemically reactive modified peptides. The functionalized bloodor blood component may then be returned to the host to provide in vivothe subject therapeutically effective conjugates. The blood also may betreated to prevent coagulation during handling ex vivo. Other sources ofblood components, such as recombinant proteins are also suitable for thepreparation of the conjugates of the present invention.

Some of the preferred compounds of the invention are derivatives ofPYY₁₋₃₆ and PYY₃₋₃₆. These derivatives comprise a strategically placedmaleimido-containing group as described above. PYY₁₋₃₆ and PYY₃₋₃₆ havethe following structures:

These peptides have an alpha helical structure starting at position 18running through to position 36 (example on PYY in Biochemistry, 2000,39, 9935). The amino acids at positions 22, 25, 29 and 33 can beconsidered as relatively important for the activity. All derivatives ofthese peptides can be truncated, modified, mutated or intact peptides.Preferably, they are able to expose side chains found on these fouramino acid residues. These residues are conserved in PYY₁₋₃₆ (SEQ ID NO:3), PYY₃₋₃₆ (SEQ ID NO: 4), pancreatic polypeptide and neuropeptide Y.Secondary conserved amino acids of potential importance can be those atpositions 5, 8, 9, 12, 15, 20, 24, 27, 32, 35 and 36.

Another aspect of the invention is to reduce excess intestinal water anddecreasing excess electrolyte secretion.

Another aspect of the invention is to relieve tumor necrosis factor(TNF)-induced acute pancreatitis through the inhibition of NF-Btranslocation to acinar nuclei (Vona-Davis L. et al., J. Am. Coll.Surg., 2004, 199, 87-95) using the DAC PYY₁₋₃₆ series of derivatives.

BRIEF DESCRIPTION OF DRAWINGS

Further features and advantages of the invention will become morereadily apparent from the following description of preferred embodimentsas illustrated by way of examples in the appended drawings wherein:

FIG. 1 is a diagram showing a comparison between the anti-obesityactivity of PYY₃₋₃₆ and the anti-obesity activity a compound accordingto a preferred embodiment of the invention, wherein the anti-obesityactivity of these compounds has been determined in an experiment byadministering them, at various doses, to Sprague-Dawley rats and bymesuring the food consumption of these rats before and afteradministration of these compounds;

FIG. 2 is another diagram as in FIG. 1, wherein the PYY peptide and thecompound according to a preferred embodiment of the invention have beenadministered to the rats according to other dosages;

FIG. 3 is a diagram showing the reduction in food intake after 24 hours,which has been generated by the administration of the PYY peptide andthe compound of the invention, during the experiment described in FIG.1;

FIG. 4 is a diagram showing the reduction in food intake after 24 hours,which has been generated by the administration of the PYY peptide andthe compound of the invention, during the experiment described in FIG.2; and

FIG. 5 is a plot showing the influence of the dosage of another coumpondaccording to a preferred embodiment of the invention on the total foodintake of Sprague-Dawley rats over time.

DESCRIPTION OF PREFERRED EMBODIMENTS

The following non-limiting examples further illustrate the invention.

Examples

1. Synthetic Scheme

General

The synthesis of the PYY peptides and functional derivatives thereof wasperformed using an automated solid-phase procedure on a Symphony PeptideSynthesizer with manual intervention during the generation of the DACpeptide. The synthesis was performed on Fmoc-protected Ramage amidelinker resin, using Fmoc-protected amino acids. Coupling was achieved byusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and diisopropylethylamine (DIEA) as theactivator cocktail in N,N-dimethylformamide (DMF) solution. The Fmocprotective group was removed using 20% piperidine/DMF. When needed, aBoc-protected amino acid was used at the N-terminus in order to generatethe free N_(α)-terminus after the peptide was cleaved from resin. Allamino acids used during the synthesis possessed the L-stereochemistryunless otherwise stated. Sigmacoted glass reaction vessels were usedduring the synthesis.

Compound I (PYY₃₋₃₆) Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp- (SEQ ID NO: 4)Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn- Leu-Val-Thr-Arg-Gln-Arg-Tyr-CONH₂

Step 1: Solid phase peptide synthesis of the DAC™ peptide on a 100 μmolescale was performed using manual and automated solid-phase synthesis, aSymphony Peptide Synthesizer and Ramage resin. The following protectedamino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Thr(tBu)-OH,Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH,Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH,Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH,Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Pro-OH,Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH,Boc-Ile-OH. They were dissolved in N,N-dimethylformamide (DMF) and,according to the sequence, activated using O-benzotriazol-1-yl-N, N, N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) andDiisopropylethylamine (DIEA). Removal of the Fmoc protecting group wasachieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1).

Step 2: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice cold(0-4° C.) Et₂O. The crude peptide was collected on a polypropylenesintered funnel, dried, redissolved in a 40% mixture of acetonitrile inwater (0.1% TFA) and lyophilized to generate the corresponding crudematerial used in the purification process.

Compound II Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp- (SEQ ID NO: 5)Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn- Leu-Val-Thr-Arg-Gln-Arg-Tyr-Lys(MPA)-CONH₂

Step 1: Solid phase peptide synthesis of the DAC derivative on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Ramage resin. The followingprotected amino acids were sequentially added to resin:Fmoc-Lys(Aloc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Leu-OH,Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH,Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH,Fmoc-Lys(Boc)-OH, Boc-Ile-OH. The following protected amino acids weresequentially added to resin: They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N, N, N′, N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).

Step 2: The selective deprotection of the Lys (Aloc) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh₃)₄ dissolved in 5 mL of C₆H₆:CHCl₃ (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl₃ (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Between every coupling, the resinwas washed 3 times with N,N-dimethylformamide (DMF) and 3 times withisopropanol.

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice cold(0-4° C.) Et₂O (Step 4). The crude peptide was collected on apolypropylene sintered funnel, dried, redissolved in a 40% mixture ofacetonitrile in water (0.1% TFA) and lyophilized to generate thecorresponding crude material used in the purification process.

Compound III MPA-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu- (SEQ ID NO: 4)Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu- Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-CONH₂

Step 1: Solid phase peptide synthesis of the DAC derivative on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Ramage resin. The followingprotected amino acids were sequentially added to resin:Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH,Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH,Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH MPA-OH. They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N, N, N′, N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).

Step 2: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice cold(0-4° C.) Et₂O (Step 4). The crude peptide was collected on apolypropylene sintered funnel, dried, redissolved in a 40% mixture ofacetonitrile in water (0.1% TFA) and lyophilized to generate thecorresponding crude material used in the purification process.

Compound IV Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp- (SEQ ID NO: 6)Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr- Tyr-Ala-Ser-Lys(MPA)-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr- CONH₂

Step 1: Solid phase peptide synthesis of the DAC derivative on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Ramage resin. The followingprotected amino acids were sequentially added to resin:Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH,Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Lys(Aloc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH,Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH,Fmoc-Lys(Boc)-OH, Boc-Ile-OH. They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N, N, N′, N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).

Step 2: The selective deprotection of the Lys (Aloc) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh₃)₄ dissolved in 5 mL of C₆H₆:CHCl₃ (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl₃ (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Between every coupling, the resinwas washed 3 times with N,N-dimethylformamide (DMF) and 3 times withisopropanol.

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (0-4° C.) (Step 4). The crude peptide was collected on apolypropylene sintered funnel, dried, redissolved in a 40% mixture ofacetonitrile in water (0.1% TFA) and lyophilized to generate thecorresponding crude material used in the purification process.

Compound V Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp- (SEQ ID NO: 7)Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg- Lys(MPA)-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg- Tyr-CONH₂

Step 1: Solid phase peptide synthesis of the DAC derivative on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Ramage resin. The followingprotected amino acids were sequentially added to resin:::Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH,Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH,Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH,Fmoc-Lys(Boc)-OH, Boc-Ile-OH They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N, N, N′, N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).

Step 2: The selective deprotection of the Lys (Aloc) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh₃)₄ dissolved in 5 mL of C₆H₆:CHCl₃ (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl₃ (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Between every coupling, the resinwas washed 3 times with N,N-dimethylformamide (DMF) and 3 times withisopropanol.

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (0-4° C.) (Step 4). The crude peptide was collected on apolypropylene sintered funnel, dried, redissolved in a 40% mixture ofacetonitrile in water (0.1% TFA) and lyophilized to generate thecorresponding crude material used in the purification process.

Compound VI Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp- (SEQ ID NO: 8)Ala-Ser-Pro-Glu-Glu-Lys(MPA)-Asn- Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr- CONH₂

Step 1: Solid phase peptide synthesis of the DAC derivative on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Ramage resin. The followingprotected amino acids were sequentially added to resin:::Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH,Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Al a-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Aloc)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH,Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH,Fmoc-Lys(Boc)-OH, Boc-Ile-OH They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N, N, N′, N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).

Step 2: The selective deprotection of the Lys (Aloc) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh₃)₄ dissolved in 5 mL of C₆H₆:CHCl₃ (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl₃ (6×5mL), 20% AcOH in DCM (6×5. mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Between every coupling, the resinwas washed 3 times with N,N-dimethylformamide (DMF) and 3 times withisopropanol.

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (0-4° C.) (Step 4). The crude peptide was collected on apolypropylene sintered funnel, dried, redissolved in a 40% mixture ofacetonitrile in water (0.1% TFA) and lyophilized to generate thecorresponding crude material used in the purification process.

Compound VII Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp- (SEQ ID NO: 9)Ala-Ser-Pro-Glu-Lys(MPA)-Leu-Asn- Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr- CONH₂

Step 1: Solid phase peptide synthesis of the DAC derivative on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Ramage resin. The followingprotected amino acids were sequentially added to resin:::Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH,Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH,Fmoc-Lys(Aloc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH,Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH,Fmoc-Lys(Boc)-OH, Boc-Ile-OH They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N, N, N′, N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).

Step 2: The selective deprotection of the Lys (Aloc) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh₃)₄ dissolved in 5 mL of C₆H₆:CHCl₃ (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl₃ (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Between every coupling, the resinwas washed 3 times with N,N-dimethylformamide (DMF) and 3 times withisopropanol.

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (0-4° C.) (Step 4). The crude peptide was collected on apolypropylene sintered funnel, dried, redissolved in a 40% mixture ofacetonitrile in water (0.1% TFA) and lyophilized to generate thecorresponding crude material used in the purification process.

Compound VIII Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu- (SEQ ID NO: 10)Asp-Ala-Ser-Pro-Lys(MPA)-Glu-Leu- Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg- Gln-Arg-Tyr-CONH₂

Step 1: Solid phase peptide synthesis of the DAC derivative on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Ramage resin. The followingprotected amino acids were sequentially added to resin:::Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH,Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH,Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH,Fmoc-Lys(Boc)-OH, Boc-Ile-OH They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N, N, N′, N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).

Step 2: The selective deprotection of the Lys (Aloc) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh₃)₄ dissolved in 5 mL of C₆H₆:CHCl₃ (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl₃ (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Between every coupling, the resinwas washed 3 times with N,N-dimethylformamide (DMF) and 3 times withisopropanol.

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (0-4° C.) (Step 4). The crude peptide was collected on apolypropylene sintered funnel, dried, redissolved in a 40% mixture ofacetonitrile in water (0.1% TFA) and lyophilized to generate thecorresponding crude material used in the purification process.

Compound IX Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu- (SEQ ID NO: 11)Asp-Ala-Ser-Lys(MPA)-Glu-Glu-Leu- Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg- Gln-Arg-Tyr-CONH₂

Step 1: Solid phase peptide synthesis of the DAC derivative on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Ramage resin. The followingprotected amino acids were sequentially added to resin:Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH,Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH,Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH,Fmoc-Lys(Boc)-OH, Boc-Ile-OH They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N, N, N′, N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).

Step 2: The selective deprotection of the Lys (Aloc) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh₃)₄ dissolved in 5 mL of C₆H₆:CHCl₃ (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl₃ (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Between every coupling, the resinwas washed 3 times with N,N-dimethylformamide (DMF) and 3 times withisopropanol.

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (0-4° C.) (Step 4). The crude peptide was collected on apolypropylene sintered funnel, dried, redissolved in a 40% mixture ofacetonitrile in water (0.1% TFA) and lyophilized to generate thecorresponding crude material used in the purification process.

Compound X Ac-Ala-Ser-Leu-Arg-His-Tyr-Leu- (SEQ ID NO: 12)Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr- CONH₂

Step 1: Solid phase peptide synthesis of the DAC derivative on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Ramage resin. The followingprotected amino acids were sequentially added to resin:Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH,Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Acetic Acid. They weredissolved in N,N-dimethylformamide (DMF) and, according to the sequence,activated using O-benzotriazol-1-yl-N, N, N′, N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).

Step 2: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (0-4° C.) (Step 4). The crude peptide was collected on apolypropylene sintered funnel, dried, redissolved in a 40% mixture ofacetonitrile in water (0.1% TFA) and lyophilized to generate thecorresponding crude material used in the purification process.

Compound XI MPA-Ala-Ser-Leu-Arg-His-Tyr-Leu- (SEQ ID NO: 12)Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr- CONH₂

Step 1: Solid phase peptide synthesis of the DAC derivative on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Ramage resin. The followingprotected amino acids were sequentially added to resin:::Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH,Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, MPA-OH. They were dissolvedin N,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N, N, N′, N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).

Step 2: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (0-4° C.) (Step 4). The crude peptide was collected on apolypropylene sintered funnel, dried, redissolved in a 40% mixture ofacetonitrile in water (0.1% TFA) and lyophilized to generate thecorresponding crude material used in the purification process.

Compound XII Ac-Ala-Ser-Leu-Arg-His-Tyr-Leu- (SEQ ID NO: 13)Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr- Lys(MPA)-CONH₂

Step 1: Solid phase peptide synthesis of the DAC derivative on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Ramage resin. The followingprotected amino acids were sequentially added to resin:Fmoc-Lys(Aloc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Leu-OH,Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, AceticAcid. They were dissolved in N,N-dimethylformamide (DMF) and, accordingto the sequence, activated using O-benzotriazol-1-yl-N, N, N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) andDiisopropylethylamine (DIEA). Removal of the Fmoc protecting group wasachieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1).

Step 2: The selective deprotection of the Lys (Aloc) group was performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh₃)₄ dissolved in 5 mL of C₆H₆:CHCl₃ (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl₃ (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

Step 3: The synthesis was then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Between every coupling, the resinwas washed 3 times with N,N-dimethylformamide (DMF) and 3 times withisopropanol.

Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (0-4° C.)

2. Purification procedure:

Each product was purified by preparative reversed phase HPLC, using aVarian (Dynamax) preparative binary HPLC system.

Purification of all the above compounds were performed using aPhenomenex Luna 10μ phenyl-hexyl, 50 mm×250 mm column (particules 10μ)equilibrated with a water/TFA mixture (0.1% TFA in H₂O; Solvent A) andacetonitrile/TFA (0.1% TFA in CH₃CN; Solvent B). Elution was achieved at50 mL/min by running various gradients of % B gradient over 180 min.Fractions containing peptide were detected by UV absorbance (VarianDynamax UVD II) at 214 and 254 nm.

Fractions were collected in 25 mL aliquots. Fractions containing thedesired product were identified by mass detection after direct injectiononto LC/MS. The selected fractions were subsequently analyzed byanalytical HPLC (20-60% B over 20 min; Phenomenex Luna 5μ phenyl-hexyl,10 mm×250 mm column, 0.5 mL/min) to identify fractions with ≦90% purityfor pooling. The pool was freeze-dried using liquid nitrogen andsubsequently lyophilized for at least 2 days to yield a white powder.

Other suitable peptides are represented in the following sequences:Tyr-Pro-Ala-Lys-Pro-Glu-Ala-Pro- (SEQ ID NO: 14)Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu- Leu-Ser-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr- Arg-Gln-Arg-Tyr; andTyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro- (SEQ ID NO: 15)Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu- Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Leu-Thr- Arg-Pro-Arg-Tyr.

3. Table of Products TABLE 1 List of the various peptides preparedtogether with their molecular weight Compound no: Theoretical M.W.Actual M.W. I 4049.5 4049.5 II 4328.8 4328.6 III 4200.6 4200.0 IV 4255.54257.1 V 4212.2 4214.1 VI 4197.2 4199.0 VII 4229.1 4231.5 VIII 4239.24241.6 IX 4255.2 4257.5 X 1931.2 1930.7 XI 2185.5 2185.0 XII 2210.52210.1

4. Flow Diagram for Each Compound:

A) Identical synthetic schemes, as exemplified in the flow diagrambelow, were employed for all stabilized DAC™. Of course, for the nativesthe Aloc removal step along with the addition step of AEEA and\or MPAwere omitted.

Direct Synthesis

Alternative Synthesis of Compound III and isolation of Compound XIII

PYY₃₋₃₆ (human) is a 34 amino acids peptide. From the sequence theN-terminal and lysine residue (in position 2) can be modified by directattachment of the DAC group. Since the peptide is not very soluble inDMF, it has to be treated with TFA to be dissolved and then neutralizedby NMM. Thus the reaction has to be in the TFA/NMM buffer system.However, both amino groups in N-terminal and lysine show the samereactivity towards MPA-OSu under the buffer system. With 1 equivalent ofMPA-OSu in the TFA/NMM system, the reaction produced four differentproducts. The differences between these products are the position of theMPA on the sequence and the number of MPA attached to the sequence. Twopositional isomers of having a single MPA group (MPA-PYY) have beenobtained as major products and two positional isomers having two MPAgroups ((MPA)₂-PYY and cyclization)) have been minor products. Thesefour products were separated by HPLC. The positional isomers bearing asingle MPA have been isolated to give MPA-PYY positional isomer-1(Compound XIII) and positional isomer-2 (Compound III) in 27.8 and 15.2%yield respectively (see the following scheme). The starting material PYYwas also recovered (37.6% recovery).

Compounds XIV and XV

In the same way, PYY can react with excess MPA-OA-OpNP for overnight togive two positional isomers having a single MPA group: MPA-OA-PYYisomer-1 (Compound XIV, 19% yield) and MPA-OA-PYY isomer-2 (Compound XV,17.2% yield). In this case, MPA-OA-OpNP ester is less reactive and thusa large excess reagent is required for the reaction to occur. The minorproducts are still cyclization and (MPA-OA)₂-PYY.

PYY (100 mg) was dissolved in DMF (5 mL) in the presence of TFA (25 μL)with the help of sonication. Then NMM (100 μL) was added followed byaddition of MPA-OSu (5.6 mg). The reaction was stirred at roomtemperature for 2.5 h. The reaction was quenched by addition of AcOH (1mL). The DMF solution was diluted with water to 20 mL. The products wereseparated by semi-preparative HPLC column (3 injections) to give PYY(37.6 mg), MPA-PYY isomer-i (Compound XII, 27.8 mg) and PMA-PYY isomer-2(Compound XIII, 15.2 mg).

PYY (50 mg) was dissolved in DMF (5 mL) in the presence of TFA (25 μL).NMM (100 μL) was then added followed by MPA-OA-OpNP (50 mg). Thereaction was stirred for 16 h at room temperature. The linker wasremoved by addition of ether and the solution removed aftercentrifugation. The precipitate was dissolved in water and injected tosemi-preparative HPLC to give MPA-OA-PYY isomer-1 (Compound XIV, 9.5 mg)and MPA-OA-PYY isomer-2 (Compound XV, 8.6 mg).

Compound XVI

Compound III was solubilized in nanopure water at a concentration of 10mM then diluted to 1 mM into a solution of HSA (25%, Cortex-Biochem, SanLeandro, Calif.). The sample were then incubated at 37° C. for 30 min.Prior to purification, the conjugate solution was diluted to 5% HSA in20 mM sodium phosphate buffer (pH 7) composed of 5 mM sodium octanoateand 750 mM (NH₄)₂SO₄.

Using an AKTA purifier (Amersham Biosciences, Uppsala, Sweden), theconjugate was loaded at a flow rate of 2.5 ml/min onto a 50 ml column ofbutyl sepharose 4 fast flow resin (Amersham Biosciences, Uppsala,Sweden) equilibrated in 20 mM sodium phosphate buffer (pH 7) composed of5 mM sodium octanoate and 750 mM (NH₄)₂SO₄. Under these conditions,Compound XVI adsorbed onto the hydrophobic resin whereas essentially allnon-conjugated (unreacted) HSA eluted within the void volume of thecolumn. The conjugate was further purified from any free (unreacted)maleimido PYY₃₋₃₆ derivative by applying a linear gradient of decreasing(NH₄)₂SO₄ concentration (750 to 0 mM) over 4 column volumes. Thepurified conjugate was then desalted and concentrated using Amicon®ultra centrifugal (30 kDa) filter devices (Millipore Corporation,Bedford, Mass.). Finally, the conjugate solution was immersed intoliquid nitrogen, lyophilized and stored at −80° C.

Compounds XVII to XXII

Compounds XVII to XXII are all conjugates having in form of a whitesolid and they have been prepared according to the same manner thanCompound XVI. The table below indicates from which peptides theseconjugates have been prepared. Moreover, the molecular weight of eachconjugate is given. TABLE 2 Conjugates obtained from various peptides.M_(r)(conjugates) Peptides Conjugate Predicted Measured Compound IIICompound XVI 70643 70639 Compound XIII Compound XXI 70645 70640 CompoundXI Compound XXII 68629 68626 Compound II Compound XIX 70771 70668Compound XII Compound XVIII 68654 68651 Compound XV Compound XVII 7078770785 Compound XIV Compound XX 70787 70785

Example I

In vitro Binding Assay: Selectivity Toward the NPY Y2 Receptor

Serially diluted test compounds (10⁻¹³M to 10⁻⁵M) were incubated for 60minutes at 37° C. in the presence of 4.09 μg of human neuropeptide Y2receptor expressing human KAN-TS cells and 50000 CPM of ¹²⁵I-PYY₃₋₃₆.The individual solutions were filtered (Whatman 934 A/H filters) andwashed with ice-cold buffer. The filters were then placed in a gammacounter and the values reported as the percent relative to the maximumgamma emission at the zero concentration as a function of test compoundconcentration as shown on Table 3. TABLE 3 Comparison of the NPY Y2Receptor binding of the PYY derivatives Peptide Sequence IC₅₀(nM)Compound I PYY₃₋₃₆ 1.17 NPY(₁₃₋₃₆) control — 2.48 Compound XVI N-term SLPYY₃₋₃₆-HSA 35.2 conjugate Compound XVII N-term LL PYY₃₋₃₆-HSA 52.4conjugate Compound XX N-K5 LL PYY₃₋₃₆-HSA 40.2 conjugate Compound XIXC-term SL PYY₃₋₃₆-HSA >10³ conjugate Compound XVIII C-term SLPYY₂₂₋₃₆-HSA >10³ conjugate

Example II

In vitro Binding Assay: Loss of Selectivity Toward the NPY Y1 Receptor

Compound I (PYY₃₋₃₆) and Compound XVI were tested so as to evaluate thepreferential binding of the Y2 receptor relative to the Y1 receptor. Aselective binding to the Y2 receptor ensures reduced (unwanted) sideeffects such as for example hypertension. TABLE 4 Comparison of the NPYY1 Receptor binding of the PYY derivatives Peptide Sequence IC₅₀(nM)Compound I PYY₃₋₃₆ 83.2 NPY(human, rat) control — 1.09 Compound XVIN-term SL PYY₃₋₃₆-HSA 875.9 conjugate

Example III

Food Intake in Rats Following IV Administration of DAC

Compound I (PYY₃₋₃₆) and Compound III were injected into the tail veinof fully grown Sprague-Dawley rats. Two experiments were carried out soas to verify the influence of the concentration on Compound III of thefood consumption of the animal. The food intake was measured pre andpost administration (see FIGS. 1 and 2). In experiment 1 on FIG. 1,4-500 g rats were used and in experiment 2 on FIG. 2, 2-300 g rats wereused.

As it can be seen from FIGS. 1 and 2, the results shown a significantreduction in food intake over the 0-12 hour and 12-24 hour periods. Theoverall effect is very significant over the 0-24 hour period at thehighest dose tested (375 nM/kg).

A comparison of reduction in food intake in the two experiments caneasily be made by using FIGS. 3 and 4. It can be seen from FIGS. 3 and 4that PYY₃₋₃₆ does not show reduction in food after 24 hrs at a dose of25 nmol/kg or at a dose of 375 nmol/kg, while Compound III, at a dose of375 nmol/kg, shows a strong effect by reducing food intake by 50% after24 hrs. This comparison demonstrates the long lasting effect of CompoundIII as compared to the free peptide PYY₃₋₃₆ in vivo.

Example IV

Peripheral vs. Central Action of Compound III

A publication by Batterham (Nature, 2002, 418, 650-654) demonstrated thestrong effect of PYY₃₋₃₆ administration into the arcuate nucleus to ratson overall food intake. The arcuate nucleus does possess a blood brainbarrier and therefore no evidence was ever shown in the literature thatperipheral neuropeptide Y2 receptors would have and influence on foodintake. Applicant has shown that Compound XVI cannot cross the bloodbrain barrier (molecular weight>70 000 Da). It is known that PYY₃₋₃₆interacts with the Y2 receptor found in the arcuate nucleus of thehypothalamus. This receptor is found behind the blood brain barrier(BBB). Nonaka et al., in an article entitled “Characterization ofblood-brain barrier permeability to PYY₃₋₃₆ in the mouse” and publishedin J. Pharmacol. Exp. Ther. 2003, 306, 948-53, have hypothesized thatthe PYY must “cross the BBB” in order to be responsible for the appetiteregulating activity.

The injection of Compound XVI i.p. into acclimatized Sprague-Dawley ratsin a repeat of the Batterham experiment showed the results shown in FIG.5.

According to FIG. 5, the 375 nmol/kg dose showed significant reductionin food intake at the 4 hour time point in the experiment. The resultsare comparable to PYY3-36 25 nmol/kg. Even though there is 15 fold moreCompound XVI, pharmacokinetics of absorption will play a role in thishead to head comparison. Compound XVI will peak in plasma at a latertime than the short peptide. This experiment was done to compare the HSAconjugate directly to the peptide.

It has thus been demonstrated from FIGS. 3 and 4 that the compounds ofthe invention are very effective for treating food disorders such asobesity. In fact the peptide (Compound III) demonstrated an activitywhich clearly superior than the activity of PYY₃₋₃₆. It can also beinferred from the results shown in FIG. 5 that the conjugate (CompoundXVI) is prevented from crossing the blood brain barrier. In factreduction in food intake via the PYY receptors (Y1 and Y2, which arethought to have an important role in appetite reduction) is thought tobe found on the arcuate nucleus. There is a blood brain barrierseparating the arcuate nucleus from plasma. The importance of thisexperiment is that the conjugate (Compound XVI) has a molecular mass >70kDA. Therefore, this compound does not cross the blood brain barrier. Itcan thus be assumed that the PYY receptors involved in the reduction offood intake are found peripherally.

As demonstrated in Kratz et al. J. Med. Chem. 2002, 45, 5523-33, when acompound containing a reactive maleimide group such as compound III isinjected in a patient, this compound will be eventually covalentlybonded to albumin, thereby being converted into Compound XVI. It canthus be said from FIGS. 1 to 5 that the enhanced activity of CompoundIII with respect to the activity of PYY₃₋₃₆ is due to the fact thatCompound III is prevented from crossing the blood brain barrier when thelatter is covalently bonded to HSA (converted into Compound XVI).

It has thus been surprisingly noted that by preventing a PYY peptides orderivative thereof from crossing the blood brain barrier, an enhancedanti-obesity activity of this peptide is observed as compared to thepeptide alone.

1. A compound comprising a peptide of formula:X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-A

wherein X₁ is absent, tyr or ala; X₂ is absent or pro; X₃ is absent, lysor an analog thereof, ile, leu, or ala; X₄ is absent, lys or an analogthereof, or glu; X₅ is absent or pro; X₆ is absent, glu, val or asp; X₇is absent, ala, tyr or asn; X₈ is absent or pro; X₉ is absent or gly;X₁₀ is absent, glu or asp; X₁₁ is absent, asp or asn; X₁₂ is absent, lysor an analog thereof, or ala; X₁₃ is absent, lys or an analog thereof,ser, thr, or pro; X₁₄ is absent, lys or an analog thereof, ala, or pro;X₁₅ is absent, lys or an analog thereof, or glu; X₁₆ is absent, glu, glnor asp; X₁₇ is absent, leu or met; X₁₈ is absent, lys or an analogthereof, ser, or ala; X₁₉ is absent, arg or gln; X₂₀ is absent or tyr;X₂₁ is absent, tyr or ala; X₂₂ is ala or ser; X₂₃ is ser, asp or ala;X₂₄ is leu; X₂₅ is arg or lys; X₂₆ is his, arg or lys; X₂₇ is tyr; X₂₈is leu or ile; X₂₉ is asn X₃₀ is leu or met; X₃₁ is val, leu or ile; X₃₂is thr; X₃₃ is arg or lys; X₃₄ is gln or pro; X₃₅ is arg or lys; X₃₆ istyr or a derivative thereof; and A is absent lys or a derivativethereof, and at least one reactive group coupled to any one of X₁ to X₃₆and A, directly or via a linking group.
 2. The compound of claim 1,wherein said compound comprises only one reactive group, said reactivegroup being coupled to any one of X₁-X₂₁, X₂₃, X₂₄, X₂₆ to X₂₈, X₃₀ toX₃₂, X₃₄ to X₃₆, and A.
 3. The compound of claim 1, wherein saidreactive group is coupled to said peptide by means of a linking group.4. The compound of claim 1, wherein X₁ is absent, said reactive group,linking group-(reactive group), tyr or ala, said tyr or ala beingoptionally coupled to said reactive group or to said linkinggroup-(reactive group); X₂ is absent, pro, said reactive group or saidlinking group-(reactive group); X₃ is absent, lys or an analog thereof,(reactive group)-lys, (reactive group)-linking group-lys, (reactivegroup)-lys analog, (reactive group)-linking group-lys analog, ile, leu,or ala, wherein said reactive group is coupled to the free amine of lysor lys analog; X₄ is absent, lys or an analog thereof, (reactivegroup)-lys, (reactive group)-linking group-lys, (reactive group)-lysanalog, (reactive group)-linking group-lys analog, or glu, wherein saidreactive group is coupled to the free amine of lys or lys analog; X₅ isabsent or pro; X₆ is absent, glu, val or asp; X₇ is absent, ala, tyr orasn; X₈ is absent or pro; X₉ is absent or gly; X₁₀ is absent, glu orasp; X₁₁ is absent, asp or asn; X₁₂ is absent, lys or an analog thereof,(reactive group)-lys, (reactive group)-linking group-lys, (reactivegroup)-lys analog, (reactive group)-linking group-lys analog, or ala,wherein said reactive group is coupled to the free amine of lys or lysanalog; X₁₃ is absent, lys or an analog thereof, (reactive group)-lys,(reactive group)-linking group-lys, (reactive group)-lys analog,(reactive group)-linking group-lys analog, ser, thr, or pro, whereinsaid reactive group is coupled to the free amine of lys or lys analog;X₁₄ is absent, lys or an analog thereof, (reactive group)-lys, (reactivegroup)-linking group-lys, (reactive group)-lys analog, (reactivegroup)-linking group-lys analog, ala or pro, wherein said reactive groupis coupled to the free amine of lys or lys analog; X₁₅ is absent, lys oran analog thereof, (reactive group)-lys, (reactive group)-linkinggroup-lys, (reactive group)-lys analog, (reactive group)-linkinggroup-lys analog, or glu, wherein said reactive group is coupled to thefree amine of lys or lys analog; X₁₆ is absent, glu, gln or asp; X₁₇ isabsent, leu or met; X₁₈ is absent, lys or an analog thereof, (reactivegroup)-lys, (reactive group)-linking group-lys, (reactive group)-lysanalog, (reactive group)-linking group-lys analog, ser, or ala, whereinsaid reactive group is coupled to the free amine of lys or lys analog;X₁₉ is absent, arg or gln; X₂₀ is absent or tyr; X₂₁ is absent, tyr,ala, a reactive group, or linking group-(reactive group), wherein saidlinking group is coupled to X₂₀ and X₂₂; X₂₂ is ala or ser; X₂₃ is ser,asp or ala; X₂₄ is leu; X₂₅ is arg or lys; X₂₆ is his, arg or lys; X₂₇is tyr; X₂₈ is leu or ile; X₂₉ is asn X₃₀ is leu or met; X₃₁ is val, leuor ile; X₃₂ is thr; X₃₃ is arg or lys; X₃₄ is gln or pro; X₃₅ is arg orlys; X₃₆ is tyr or a derivative thereof; A is absent, lys or aderivative thereof, (reactive group)-lys, (reactive group)-linkinggroup-lys, (reactive group)-lys derivative, (reactive group)-linkinggroup-lys derivative, wherein said reactive group is coupled to the freeamine of lys or lys derivative; and wherein said compound comprises onlyone reactive group.
 5. The compound of claim 4, wherein X₁ is absent; X₂is absent; X₃ leu; X₄ is lys, (reactive group)-lys or (reactivegroup)-linking group-lys; X₅ is pro; X₆ is glu; X₇ is ala; X₈ is pro; X₉is gly; X₁₀ is glu; X₁₁ is asp; X₁₂ is ala; X₁₃ is ser; X₁₄ is pro; X₁₅is glu; X₁₆ is glu; X₁₇ is leu; X₁₈ is ser; X₁₉ is arg; X₂₀ is tyr; X₂₁is tyr; X₂₂ is ala; X₂₃ is ser; X₂₄ is leu; X₂₅ is arg; X₂₆ is his; X₂₇is tyr; X₂₈ is leu; X₂₉ is asn; X₃₀ is leu; X₃₁ is val; X₃₂ is thr; X₃₃is arg; X₃₄ is gln; X₃₅ is arg; X₃₆ is tyr or a derivative thereof; A isabsent, lys or a derivative thereof, (reactive group)-lys, (reactivegroup)-linking group-lys, (reactive group)-lys derivative, (reactivegroup)-linking group-lys derivative, wherein said reactive group iscoupled to the free amine of lys or lys derivative.
 6. The compound ofclaim 1, wherein said reactive group is a Michael acceptor, asuccinimidyl-containing group, a maleimido-containing group or anelectrophilic thiol acceptor.
 7. The compound of claim 1, wherein saidreactive group is a maleimido-containing group.
 8. The compound of claim1, wherein said reactive group is capable of reacting with an aminogroup, a hydroxyl group or a thiol group on a blood component so as toform a stable covalent bond.
 9. The compound of claim 8, wherein saidblood component is a protein.
 10. The compound of claim 9, wherein saidprotein is albumin
 11. The compound of claim 3, wherein said linkinggroup is selected from the group consisting of (2-amino) ethoxy aceticacid (AEA), ethylenediamine (EDA), amino ethoxy ethoxy succinimic acid(AEES), 2-[2-(2-amino)ethoxy)] ethoxy acetic acid (AEEA), AEEA-AEEA,—NH₂—(CH₂)_(n)-COOH where n is an integer from 1 to 20; one or morealkyl chains (C₁-C₁₀) saturated or unsaturated which optionallycomprises oxygen, nitrogen or sulfur atoms motifs, glycine,3-aminopropionic acid (APA), 8-aminooctanoic acid (OA), 4-aminobenzoicacid (APhA), and combinations thereof.
 12. The compound of claim 1,wherein said peptide is a peptide as defined in any one of SEQ IDS NO: 2to
 13. 13. The compound of claim 1, wherein said peptide is a peptide ofSEQ ID NO:
 4. 14. The compound of claim 1, wherein the tyr derivative isof formula

where R₁ is H, a protecting group (PG), a C₁-C₁₀ branched, linear orcyclic alkyl, a phosphate or a sulfate; R₂ and R₃ are same or differentand selected from the group consisting of H, D and I; R₄ is OH, OPG,OR₅, SH, SPG, SR₅, NH₂, NHPG, N(PG)₂, N(R₅)₂, NR₅PG, or NHR₆, where R₅is a C₁-C₁₀ branched, linear or cyclic alkyl, and R₆ is a solid phasesupport, and wherein the lys derivative is of formula:

where R₄ is as previously defined; and n is an integer having a value of0, 1, 2, 3 or
 4. 15. The compound of claim 1, wherein the lys analog isof formula:

where n is an integer having a value of 0, 1, 2, 3 or
 4. 16. A conjugatecomprising a blood component and a compound having a peptide of formula:X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-A

wherein X₁ is absent, tyr or ala; X₂ is absent or pro; X₃ is absent, lysor an analog thereof, ile, leu, or ala; X₄ is absent, lys or an analogthereof, or glu; X₅ is absent or pro; X₆ is absent, glu, val or asp; X₇is absent, ala, tyr or asn; X₈ is absent or pro; X₉ is absent or gly;X₁₀ is absent, glu or asp; X₁₁ is absent, asp or asn; X₁₂ is absent, lysor an analog thereof, or ala; X₁₃ is absent, lys or an analog thereof,ser, thr, or pro; X₁₄ is absent, lys or an analog thereof, ala, or pro;X₁₅ is absent, lys or an analog thereof, or glu; X₁₆ is absent, glu, glnor asp; X₁₇ is absent, leu or met; X₁₈ is absent, lys or an analogthereof, ser, or ala; X₁₉ is absent, arg or gln; X₂₀ is absent or tyr;X₂₁ is absent, tyr or ala; X₂₂ is ala or ser; X₂₃ is ser, asp or ala;X₂₄ is leu; X₂₅ is arg or lys; X₂₆ is his, arg or lys; X₂₇ is tyr; X₂₈is leu or ile; X₂₉ is asn X₃₀ is leu or met; X₃₁ is val, leu or ile; X₃₂is thr; X₃₃ is arg or lys; X₃₄ is gln or pro; X₃₅ is arg or lys; X₃₆ istyr or a derivative thereof; and A is absent, lys or a derivativethereof, and a reactive group coupled to any one of X₁-X₃₆ and A,directly or via a linking group, and wherein said reactive group iscoupled with at least an amino group, a hydroxyl group or a thiol groupon said blood component so as to form a stable covalent bond therewith.17. The conjugate of claim 16, wherein said reactive group is coupled toany one of X₁-X₂₁, X₂₃, X₂₄, X₂₆ to X₂₈, X₃₀ to X₃₂, X₃₄ to X₃₆, and A.18. The conjugate of claim 16, wherein said reactive group is coupled tosaid peptide by means of a linking group.
 19. The conjugate of claim 16,wherein X₁ is absent, said reactive group, linking group-(reactivegroup), tyr or ala, said tyr or ala being optionally coupled to saidreactive group or to said linking group-(reactive group); X₂ is absent,pro, said reactive group or said linking group-(reactive group); X₃ isabsent, lys or an analog thereof, (reactive group)-lys, (reactivegroup)-linking group-lys, (reactive group)-lys analog, (reactivegroup)-linking group-lys analog, ile, leu, or ala, wherein-said reactivegroup is coupled to the free amine of lys or lys analog; X₄ is absent,lys or an analog thereof, (reactive group)-lys, (reactive group)-linkinggroup-lys, (reactive group)-lys analog, (reactive group)-linkinggroup-lys analog, or glu, wherein said reactive group is coupled to thefree amine of lys or lys analog; X₅ is absent or pro; X₆ is absent, glu,val or asp; X₇ is absent, ala, tyr or asn; X₈ is absent or pro; X₉ isabsent or gly; X₁₀ is absent, glu or asp; X₁₁ is absent, asp or asn; X₁₂is absent, lys or an analog thereof, (reactive group)-lys, (reactivegroup)-linking group-lys, (reactive group)-lys analog, (reactivegroup)-linking group-lys analog, or ala, wherein said reactive group iscoupled to the free amine of lys or lys analog; X₁₃ is absent, lys or ananalog thereof, (reactive group)-lys, (reactive group)-linkinggroup-lys, (reactive group)-lys analog, (reactive group)-linkinggroup-lys analog, ser, thr, or pro, wherein said reactive group iscoupled to the free amine of lys or lys analog; X₁₄ is absent, lys or ananalog thereof, (reactive group)-lys, (reactive group)-linkinggroup-lys, (reactive group)-lys analog, (reactive group)-linkinggroup-lys analog, ala or pro, wherein said reactive group is coupled tothe free amine of lys or lys analog; X₁₅ is absent, lys or an analogthereof, (reactive group)-lys, (reactive group)-linking group-lys,(reactive group)-lys analog, (reactive group)-linking group-lys analog,or glu, wherein said reactive group is coupled to the free amine of lysor lys analog; X₁₆ is absent, glu, gln or asp; X₁₇ is absent, leu ormet; X₁₈ is absent, lys or an analog thereof, (reactive group)-lys,(reactive group)-linking group-lys, (reactive group)-lys analog,(reactive group)-linking group-lys analog, ser, or ala, wherein saidreactive group is coupled to the free amine of lys or lys analog; X₁₉ isabsent, arg or gln; X₂₀ is absent or tyr; X₂₁ is absent, tyr, ala, areactive group, or linking group-(reactive group), wherein said linkinggroup is coupled to X₂₀ and X₂₂; X₂₂ is ala or ser; X₂₃ is ser, asp orala; X₂₄ is leu; X₂₅ is arg or lys; X₂₆ is his, arg or lys; X₂₇ is tyr;X₂₈ is leu or ile; X₂₉ is asn X₃₀ is leu or met; X₃₁ is val, leu or ile;X₃₂ is thr; X₃₃ is arg or lys; X₃₄ is gln or pro; X₃₅ is arg or lys; X₃₆is tyr or a derivative thereof; A is absent, lys or a derivativethereof, (reactive group)-lys, (reactive group)-linking group-lys,(reactive group)-lys derivative, (reactive group)-linking group-lysderivative, wherein said reactive group is coupled to the free amine oflys or lys derivative; and wherein said conjugate comprises only onereactive group.
 20. The conjugate of claim 19, wherein X₁ is absent; X₂is absent; X₃ leu; X₄ is lys, (reactive group)-lys or (reactivegroup)-linking group-lys; X₅ is pro; X₆ is glu; X₇ is ala; X₈ is pro; X₉is gly; X₁₀ is glu; X₁₁ is asp; X₁₂ is ala; X₁₃ is ser; X₁₄ is pro; X₁₅is glu; X₁₆ is glu; X₁₇ is leu; X₁₈ is ser; X₁₉ is arg; X₂₀ is tyr; X₂₁is tyr; X₂₂ is ala; X₂₃ is ser; X₂₄ is leu; X₂₅ is arg; X₂₆ is his; X₂₇is tyr; X₂₈ is leu; X₂₉ is asn; X₃₀ is leu; X₃₁ is val; X₃₂ is thr; X₃₃is arg; X₃₄ is gln; X₃₅ is arg; X₃₆ is tyr or a derivative thereof; A isabsent, lys or a derivative thereof, (reactive group)-lys, (reactivegroup)-linking group-lys, (reactive group)-lys derivative, (reactivegroup)-linking group-lys derivative, where said reactive group iscoupled to the free amine of lys or lys derivative.
 21. The conjugate ofclaim 16, wherein said reactive group is a Michael acceptor, asuccinimidyl-containing group, a maleimido-containing group or anelectrophilic thiol acceptor.
 22. The conjugate of claim 16, whereinsaid reactive group is a maleimido-containing group.
 23. The conjugateof claim 16, wherein said blood component is a protein.
 24. Theconjugate of claim 23, wherein said protein is albumin
 25. The conjugateof claim 18, wherein said linking group is selected from the groupconsisting of (2-amino) ethoxy acetic acid (AEA), ethylenediamine (EDA),amino ethoxy ethoxy succinimic acid (AEES), 2-[2-(2-amino)ethoxy)]ethoxy acetic acid (AEEA), AEEA-AEEA, —NH₂—(CH₂)_(n)-COOH where n is aninteger from 1 to 20; one or more alkyl chains (C₁-C₁₀) saturated orunsaturated which optionally comprises oxygen, nitrogen or sulfur atomsmotifs, glycine, 3-aminopropionic acid (APA), 8-aminooctanoic acid (OA),4-aminobenzoic acid (APhA), and combinations thereof.
 26. The conjugateof claim 16, wherein said peptide is a peptide as defined in any one ofSEQ IDS NO: 2 to
 13. 27. The conjugate of claim 16, wherein said peptideis a peptide of SEQ ID NO:
 4. 28. The conjugate of claim 16, wherein thetyr derivative is of formula

where R₁ is H, a protecting group (PG), a C₁-C₁₀ branched, linear orcyclic alkyl, a phosphate or a sulfate; R₂ and R₃ are same or differentand selected from the group consisting of H, D and I; R₄ is OH, OPG,OR₅, SH, SPG, SR₅, NH₂, NHPG, N(PG)₂, N(R₅)₂, NR₅PG, or NHR₆, where R₅is a C₁-C₁₀ branched, linear or cyclic alkyl, and R₆ is a solid phasesupport, and wherein the lys derivative is of formula:

where R₄ is as previously defined; and n is an integer having a value of0, 1, 2, 3 or
 4. 29. The conjugate of claim 16, wherein the lys analogis of formula:

where n is an integer having a value of 0, 1, 2, 3 or
 4. 30. A compoundcomprising a PYY peptide or a functional derivative thereof which iscoupled to a reactive group, said reactive group being capable ofreacting with an amino group, a hydroxyl group or a thiol group on ablood component so as to form a stable covalent bond therewith, therebysubstantially preventing said PYY peptide or functional derivativethereof from crossing the blood brain barrier.
 31. The compound of claim30, wherein said PYY peptide comprises PYY-₁₋₃₆ or PYY₃₋₃₆.
 32. Thecompound of claim 30, wherein said PYY peptide or said functionalderivative thereof is coupled to said reactive group by means of alinking group.
 33. The compound of claim 30, wherein said PYY functionalderivative comprises a peptide of formula:Z₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀-Z₁₁-Z₁₂ wherein Z₁ is ala; Z₄ is arg; Z₈is asn; Z₁₂ is arg; and Z₂, Z₃, Z₅ to Z₇ and Z₉ to Z₁₁ are selected fromthe group consisting of the natural amino acids, said reactive groupbeing coupled to any one of Z₁-Z₁₂.
 34. The compound of claim 33,wherein said reactive group is coupled to any one Z₂, Z₃, Z₅ to Z₇ andZ₉ to Z₁₁.
 35. The compound of claim 30, wherein said PYY functionalderivative comprises a peptide of formula:X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-A

wherein X₁ is absent, tyr or ala; X₂ is absent or pro; X₃ is absent, lysor an analog thereof, ile, leu, or ala; X₄ is absent, lys or an analogthereof, or glu; X₅ is absent or pro; X₆ is absent, glu, val or asp; X₇is absent, ala, tyr or asn; X₈ is absent or pro; X₉ is absent or gly;X₁₀ is absent, glu or asp; X₁₁ is absent, asp or asn; X₁₂ is absent, lysor an analog thereof, or ala; X₁₃ is absent, lys or an analog thereof,ser, thr, or pro; X₁₄ is absent, lys or an analog thereof, ala, or pro;X₁₅ is absent, lys or an analog thereof, or glu; X₁₆ is absent, glu, glnor asp; X₁₇ is absent, leu or met; X₁₈ is absent, lys or an analogthereof, ser, or ala; X₁₉ is absent, arg or gln; X₂₀ is absent or tyr;X₂₁ is absent, tyr or ala; X₂₂ is ala or ser; X₂₃ is ser, asp or ala;X₂₄ is leu; X₂₅ is arg or lys; X₂₆ is his, arg or lys; X₂₇ is tyr; X₂₈is leu or ile; X₂₉ is asn X₃₀ is leu or met; X₃₁ is val, leu or ile; X₃₂is thr; X₃₃ is arg or lys; X₃₄ is gln or pro; X₃₅ is arg or lys; X₃₆ istyr or a derivative thereof; and A is absent lys or a derivativethereof, said reactive being group coupled to any one of X₁-X₃₆ and A.36. The compound of claim 35, wherein said reactive group is coupled toany one of X₁-X₂₁, X₂₃, X₂₄, X₂₆ to X₂₈, X₃₀ to X₃₂, X₃₄ to X₃₆, and A.37. The compound of claim 35, wherein said reactive group is coupled tosaid peptide or said derivative by means of a linking group.
 38. Thecompound of claim 35, wherein X₁ is absent, said reactive group, linkinggroup-(reactive group), tyr or ala, said tyr or ala being optionallycoupled to said reactive group or to said linking group-(reactivegroup); X₂ is absent, pro, said reactive group or said linkinggroup-(reactive group); X₃ is absent, lys or an analog thereof,(reactive group)-lys, (reactive group)-linking group-lys, (reactivegroup)-lys analog, (reactive group)-linking group-lys analog, ile, leu,or ala, wherein said reactive group is coupled to the free amine of lysor lys analog; X₄ is absent, lys or an analog thereof, (reactivegroup)-lys, (reactive group)-linking group-lys, (reactive group)-lysanalog, (reactive group)-linking group-lys analog, or glu, wherein saidreactive group is coupled to the free amine of lys or lys analog; X₅ isabsent or pro; X₆ is absent, glu, val or asp; X₇ is absent, ala, tyr orasn; X₈ is absent or pro; X₉ is absent or gly; X₁₀ is absent, glu orasp; X₁₁ is absent, asp or asn; X₁₂ is absent, lys or an analog thereof,(reactive group)-lys, (reactive group)-linking group-lys, (reactivegroup)-lys analog, (reactive group)-linking group-lys analog, or ala,wherein said reactive group is coupled to the free amine of lys or lysanalog; X₁₃ is absent, lys or an analog thereof, (reactive group)-lys,(reactive group)-linking group-lys, (reactive group)-lys analog,(reactive group)-linking group-lys analog, ser, thr, or pro, whereinsaid reactive group is coupled to the free amine of lys or lys analog;X₁₄ is absent, lys or an analog thereof, (reactive group)-lys, (reactivegroup)-linking group-lys, (reactive group)-lys analog, (reactivegroup)-linking group-lys analog, ala or pro, wherein said reactive groupis coupled to the free amine of lys or lys analog; X₁₅ is absent, lys oran analog thereof, (reactive group)-lys, (reactive group)-linkinggroup-lys, (reactive group)-lys analog, (reactive group)-linkinggroup-lys analog, or glu, wherein said reactive group is coupled to thefree amine of lys or lys analog; X₁₆ is absent, glu, gln or asp; X₁₇ isabsent, leu or met; X₁₈ is absent, lys or an analog thereof, (reactivegroup)-lys, (reactive group)-linking group-lys, (reactive group)-lysanalog, (reactive group)-linking group-lys analog, ser, or ala, whereinsaid reactive group is coupled to the free amine of lys or lys analog;X₁₉ is absent, arg or gln; X₂₀ is absent or tyr; X₂₁ is absent, tyr,ala, a reactive group, or linking group-(reactive group), wherein saidlinking group is coupled to X₂₀ and X₂₂; X₂₂ is ala or ser; X₂₃ is ser,asp or ala; X₂₄ is leu; X₂₅ is arg or lys; X₂₆ is his, arg or lys; X₂₇is tyr; X₂₈ is leu or ile; X₂₉ is asn X₃₀ is leu or met; X₃₁ is val, leuor ile; X₃₂ is thr; X₃₃ is arg or lys; X₃₄ is gln or pro; X₃₅ is arg orlys; X₃₆ is tyr or a derivative thereof; and A is absent, lys or aderivative thereof, (reactive group)-lys, (reactive group)-linkinggroup-lys, (reactive group)-lys derivative, (reactive group)-linkinggroup-lys derivative, wherein said reactive group is coupled to the freeamine of lys or lys derivative.
 39. The compound of claim 35, wherein X₁is absent; X₂ is absent; X₃ leu; X₄ is lys, (reactive group)-lys or(reactive group)-linking group-lys; X₅ is pro; X₆ is glu; X₇ is ala; X₈is pro; X₉ is gly; X₁₀ is glu; X₁₁ is asp; X₁₂ is ala; X₁₃ is ser; X₁₄is pro; X₁₅ is glu; X₁₆ is glu; X₁₇ is leu; X₁₈ is ser; X₁₉ is arg; X₂₀is tyr; X₂₁ is tyr; X₂₂ is ala; X₂₃ is ser; X₂₄ is leu; X₂₅ is arg; X₂₆is his; X₂₇ is tyr; X₂₈ is leu; X₂₉ is asn; X₃₀ is leu; X₃₁ is val; X₃₂is thr; X₃₃ is arg; X₃₄ is gln; X₃₅ is arg; X₃₆ is tyr or a derivativethereof; A is absent, lys or a derivative thereof, (reactive group)-lys,(reactive group)-linking group-lys, (reactive group)-lys derivative,(reactive group)-linking group-lys derivative, where said reactive groupis coupled to the free amine of lys or lys derivative.
 40. The compoundof claim 30, wherein said reactive group is a Michael acceptor, asuccinimidyl-containing group, a maleimido-containing group or anelectrophilic thiol acceptor.
 41. The compound of claim 30, wherein saidreactive group is a maleimido-containing group.
 42. The compound ofclaim 30, wherein said blood component is a protein.
 43. The compound ofclaim 42, wherein said protein is albumin
 44. The compound of claim 37,wherein said linking group is selected from the group consisting of(2-amino) ethoxy acetic acid (AEA), ethylenediamine (EDA), amino ethoxyethoxy succinimic acid (AEES), 2-[2-(2-amino)ethoxy)] ethoxy acetic acid(AEEA), AEEA-AEEA, —NH₂—(CH₂)_(n)-COOH where n is an integer from 1 to20; one or more alkyl chains (C₁-C₁₀) saturated or unsaturated whichoptionally comprises oxygen, nitrogen or sulfur atoms motifs, glycine,3-aminopropionic acid (APA), 8-aminooctanoic acid (OA), 4-aminobenzoicacid (APhA), and combinations thereof.
 45. The compound of claim 30,comprising a peptide as defined in any one of SEQ IDS NO: 2 to
 13. 46.The compound of claim 30, comprising a peptide of SEQ ID NO:
 4. 47. Thecompound of claim 35, wherein the tyr derivative is of formula:

where R₁ is H, a protecting group (PG), a C₁-C₁₀ branched, linear orcyclic alkyl, a phosphate or a sulfate; R₂ and R₃ are same or differentand selected from the group consisting of H, D and I; R₄ is OH, OPG,OR₅, SH, SPG, SR₅, NH₂, NHPG, N(PG)₂, N(R₅)₂, NR₅PG, or NHR₆, where R₅is a C₁-C₁₀ branched, linear or cyclic alkyl, and R₆ is a solid phasesupport, and wherein the lys derivative is of formula:

where R₄ is as previously defined; and n is an integer having a value of0, 1, 2, 3 or
 4. 48. The compound of claim 35, wherein the lys analog isof formula:

where n is an integer having a value of 0, 1, 2, 3 or
 4. 49. A conjugatecomprising a blood component; and a PYY peptide or a functionalderivative thereof which is coupled to a reactive group, wherein saidreactive group is coupled with at least an amino group, a hydroxyl groupor a thiol group on said blood component so as to form a stable covalentbond therewith, thereby substantially preventing said PYY peptide orderivative thereof from crossing the blood brain barrier.
 50. Theconjugate of claim 49, wherein said conjugate further comprises alinking group disposed between said reactive group and said PYY peptideor derivative thereof.
 51. The conjugate of claim 50, wherein saidlinking group is selected from the group consisting of (2-amino) ethoxyacetic acid (AEA), ethylenediamine (EDA), amino ethoxy ethoxy succinimicacid (AEES), 2-[2-(2-amino)ethoxy)] ethoxy acetic acid (AEEA),AEEA-AEEA, —NH₂—(CH₂)_(n)-COOH where n is an integer from 1 to 20; oneor more alkyl chains (C₁-C₁₀) saturated or unsaturated which optionallycomprises oxygen, nitrogen or sulfur atoms motifs, glycine,3-aminopropionic acid (APA), 8-aminooctanoic acid (OA), 4-aminobenzoicacid (APhA), and combinations thereof.
 52. The conjugate of claim 49,wherein said blood component is a blood protein.
 53. The conjugate ofclaim 52, wherein said blood protein is albumin.
 54. The conjugate ofclaim 49, wherein said PYY peptide comprises PYY₁₋₃₆ or PYY₃₋₃₆.
 55. Theconjugate of claim 49, wherein said conjugate comprises said PYYfunctional derivative.
 56. The conjugate of claim 55, wherein saidfunctional derivative comprises a peptide of formula:Z₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀-Z₁₁-Z₁₂ wherein Z₁ is ala or ser; Z₄ isarg; Z₈ is asn; Z₁₂ is arg; and Z₂, Z₃, Z₅ to Z₇ and Z₉ to Z₁₁ areselected from the group consisting of the natural amino acids, saidreactive group being coupled to any one of Z₁-Z₁₂.
 57. The conjugate ofclaim 56, wherein said reactive group is coupled to any one Z₂, Z₃, Z₅to Z₇ and Z₉ to Z₁₁.
 58. The conjugate of claim 49 wherein said PYYfunctional derivative comprises a peptide of formula:X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-A

wherein X₁ is absent, tyr or ala; X₂ is absent or pro; X₃ is absent, lysor an analog thereof, ile, leu, or ala; X₄ is absent, lys or an analogthereof, or glu; X₅ is absent or pro; X₆ is absent, glu, val or asp; X₇is absent, ala, tyr or asn; X₈ is absent or pro; X₉ is absent or gly;X₁₀ is absent, glu or asp; X₁₁ is absent, asp or asn; X₁₂ is absent, lysor an analog thereof, or ala; X₁₃ is absent, lys or an analog thereof,ser, thr, or pro; X₁₄ is absent, lys or an analog thereof, ala, or pro;X₁₅ is absent, lys or an analog thereof, or glu; X₁₆ is absent, glu, glnor asp; X₁₇ is absent, leu or met; X₁₈ is absent, lys or an analogthereof, ser, or ala; X₁₉ is absent, arg or gln; X₂₀ is absent or tyr;X₂₁ is absent, tyr or ala; X₂₂ is ala or ser; X₂₃ is ser, asp or ala;X₂₄ is leu; X₂₅ is arg or lys; X₂₆ is his, arg or lys; X₂₇ is tyr; X₂₈is leu or ile; X₂₉ is asn X₃₀ is leu or met; X₃₁ is val, leu or ile; X₃₂is thr; X₃₃ is arg or lys; X₃₄ is gln or pro; X₃₅ is arg or lys; X₃₆ istyr or a derivative thereof; and A is absent lys or a derivativethereof, and said reactive group is coupled to any one of X₁-X₃₆ and A,59. The conjugate of claim 58, wherein said reactive group is coupled toany one of X₁-X₂₁, X₂₃, X₂₄, X₂₆ to X₂₈, X₃₀ to X₃₂, X₃₄ to X₃₆, and A.60. The conjugate of claim 58, wherein X₁ is absent, said reactivegroup, linking group-(reactive group), tyr or ala, said tyr or ala beingoptionally coupled to said reactive group or to said linkinggroup-(reactive group); X₂ is absent, pro, said reactive group or saidlinking group-(reactive group); X₃ is absent, lys or an analog thereof,(reactive group)-lys, (reactive group)-linking group-lys, (reactivegroup)-lys analog, (reactive group)-linking group-lys analog, ile, leu,or ala, wherein said reactive group is coupled to the free amine of lysor lys analog; X₄ is absent, lys or an analog thereof, (reactivegroup)-lys, (reactive group)-linking group-lys, (reactive group)-lysanalog, (reactive group)-linking group-lys analog, or glu, wherein saidreactive group is coupled to the free amine of lys or lys analog; X₅ isabsent or pro; X₆ is absent, glu, val or asp; X₇ is absent, ala, tyr orasn; X₈ is absent or pro; X₉ is absent or gly; X₁₀ is absent, glu orasp; X₁₁ is absent, asp or asn; X₁₂ is absent, lys or an analog thereof,(reactive group)-lys, (reactive group)-linking group-lys, (reactivegroup)-lys analog, (reactive group)-linking group-lys analog, or ala,wherein said reactive group is coupled to the free amine of lys or lysanalog; X₁₃ is absent, lys or an analog thereof, (reactive group)-lys,(reactive group)-linking group-lys, (reactive group)-lys analog,(reactive group)-linking group-lys analog, ser, thr, or pro, whereinsaid reactive group is coupled to the free amine of lys or lys analog;X₁₄ is absent, lys or an analog thereof, (reactive group)-lys, (reactivegroup)-linking group-lys, (reactive group)-lys analog, (reactivegroup)-linking group-lys analog, ala or pro, wherein said reactive groupis coupled to the free amine of lys or lys analog; X₁₅ is absent, lys oran analog thereof, (reactive group)-lys, (reactive group)-linkinggroup-lys, (reactive group)-lys analog, (reactive group)-linkinggroup-lys analog, or glu, wherein said reactive group is coupled to thefree amine of lys or lys analog; X₁₆ is absent, glu, gln or asp; X₁₇ isabsent, leu or met; X₁₈ is absent, lys or an analog thereof, (reactivegroup)-lys, (reactive group)-linking group-lys, (reactive group)-lysanalog, (reactive group)-linking group-lys analog, ser, or ala, whereinsaid reactive group is coupled to the free amine of lys or lys analog;X₁₉ is absent, arg or gln; X₂₀ is absent or tyr; X₂₁ is absent, tyr,ala, a reactive group, or linking group-(reactive group), wherein saidlinking group is coupled to X₂₀ and X₂₂; X₂₂ is ala or ser; X₂₃ is ser,asp or ala; X₂₄ is leu; X₂₅ is arg or lys; X₂₆ is his, arg or lys; X₂₇is tyr; X₂₈ is leu or ile; X₂₉ is asn X₃₀ is leu or met; X₃₁ is val, leuor ile; X₃₂ is thr; X₃₃ is arg or lys; X₃₄ is gln or pro; X₃₅ is arg orlys; X₃₆ is tyr or a derivative thereof; and A is absent, lys or aderivative thereof, (reactive group)-lys, (reactive group)-linkinggroup-lys, (reactive group)-lys derivative, (reactive group)-linkinggroup-lys derivative, where said reactive group is coupled to the freeamine of lys or lys derivative.
 61. The conjugate of claim 60, whereinX₁ is absent; X₂ is absent; X₃ leu; X₄ is lys, (reactive group)-lys or(reactive group)-linking group-lys; X₅ is pro; X₆ is glu; X₇ is ala; X₈is pro; X₉ is gly; X₁₀ is glu; X₁₁ is asp; X₁₂ is ala; X₁₃ is ser; X₁₄is pro; X₁₅ is glu; X₁₆ is glu; X₁₇ is leu; X₁₈ is ser; X₁₉ is arg; X₂₀is tyr; X₂₁ is tyr; X₂₂ is ala; X₂₃ is ser; X₂₄ is leu; X₂₅ is arg; X₂₆is his; X₂₇ is tyr; X₂₈ is leu; X₂₉ is asn; X₃₀ is leu; X₃₁ is val; X₃₂is thr; X₃₃ is arg; X₃₄ is gln; X₃₅ is arg; X₃₆ is tyr or a derivativethereof; A is absent, lys or a derivative thereof, (reactive group)-lys,(reactive group)-linking group-lys, (reactive group)-lys derivative,(reactive group)-linking group-lys derivative, where said reactive groupis coupled to the free amine of lys or lys derivative.
 62. The conjugateof claim 49, wherein said reactive group is a Michael acceptor, asuccinimidyl-containing group, a maleimido-containing group or anelectrophilic thiol acceptor.
 63. The conjugate of claim 49, whereinsaid reactive group is a maleimido-containing group.
 64. The conjugateof claim 49, comprising a peptide as defined in any one of SEQ IDS NO: 2to
 13. 65. The conjugate of claim 49, comprising a peptide of SEQ ID NO:4.
 66. The conjugate of claim 58, wherein the tyr derivative is offormula

where R₁ is H, a protecting group (PG), a C₁-C₁₀ branched, linear orcyclic alkyl, a phosphate or a sulfate; R₂ and R₃ are same or differentand selected from the group consisting of H, D and I; R₄ is OH, OPG,OR₅, SH, SPG, SR₅, NH₂, NHPG, N(PG)₂, N(R₅)₂, NR₅PG, or NHR₆, where R₅is a C₁-C₁₀ branched, linear or cyclic alkyl, and R₆ is a solid phasesupport, and wherein the lys derivative is of formula:

where R₄ is as previously defined; and n is an integer having a value of0, 1, 2, 3 or
 4. 67. The conjugate of claim 58, wherein the lys analogis of formula:

where n is an integer having a value of 0, 1, 2, 3 or
 4. 68. Theconjugate of claim 49, wherein said PYY peptide is PYY₁₋₃₆, or PYY₃₋₃₆.69. A method of enhancing the anti-obesity activity of a PYY peptide orfunctional derivative thereof comprising the step of covalently bondingsaid PYY peptide or functional derivative thereof to a blood component,thereby preventing said PYY peptide or functional derivative thereoffrom crossing the blood brain barrier when administered to said patient,whereby preventing said PYY peptide or functional derivative thereoffrom crossing the blood brain barrier results in an enhancedanti-obesity activity of said PYY peptide or functional derivativethereof.
 70. The method of claim 69, wherein said PYY peptide comprisesPYY₁₋₃₆ or PYY₃₋₃₆.
 71. The method of claim 69, wherein in said methodthe PYY functional derivative is covalently bonded to the bloodcomponent.
 72. The method of claim 69, wherein said PYY peptide orfunctional derivative thereof is coupled to a reactive group which iscapable of reacting with an amino group, a hydroxyl group or a thiolgroup on said blood component so as to form a stable covalent bondtherewith.
 73. The method of claim 71, wherein said PYY functionalderivative comprises a peptide of formula:Z₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀-Z₁₁-Z₁₂ wherein Z₁ is ala or ser; Z₄ isarg; Z₈ is asn; Z₁₂ is arg; and Z₂, Z₃, Z₅ to Z₇ and Z₉ to Z₁₁ areselected from the group consisting of the natural amino acids, saidreactive group being coupled to any one of Z₁₁-Z₁₂.
 74. The method ofclaim 73, wherein said reactive group is coupled to any one Z₂, Z₃, Z₅to Z₇ and Z₉ to Z₁₁.
 75. The method of claim 72, wherein said PYYfunctional derivative comprises a peptide of formula:X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃-X₂₄-X₂₅-X₂₆-X₂₇-X₂₈-X₂₉-X₃₀-X₃₁-X₃₂-X₃₃-X₃₄-X₃₅-X₃₆-A

wherein X₁ is absent, tyr or ala; X₂ is absent or pro; X₃ is absent, lysor an analog thereof, ile, leu, or ala; X₄ is absent, lys or an analogthereof, or glu; X₅ is absent or pro; X₆ is absent, glu, val or asp; X₇is absent, ala, tyr or asn; X₈ is absent or pro; X₉ is absent or gly;X₁₀ is absent, glu or asp; X₁₁ is absent, asp or asn; X₁₂ is absent, lysor an analog thereof, or ala; X₁₃ is absent, lys or an analog thereof,ser, thr, or pro; X₁₄ is absent, lys or an analog thereof, ala, or pro;X₁₅ is absent, lys or an analog thereof, or glu; X₁₆ is absent, glu, glnor asp; X₁₇ is absent, leu or met; X₁₈ is absent, lys or an analogthereof, ser, or ala; X₁₉ is absent, arg or gln; X₂₀ is absent or tyr;X₂₁ is absent, tyr or ala; X₂₂ is ala or ser; X₂₃ is ser, asp or ala;X₂₄ is leu; X₂₅ is arg or lys; X₂₆ is his, arg or lys; X₂₇ is tyr; X₂₈is leu or ile; X₂₉ is asn X₃₀ is leu or met; X₃₁ is val, leu or ile; X₃₂is thr; X₃₃ is arg or lys; X₃₄ is gln or pro; X₃₅ is arg or lys; X₃₆ istyr or a derivative thereof; and A is absent lys or a derivativethereof, said reactive being group coupled to any one of X₁-X₃₆ and A.76. The method of claim 75, wherein said reactive group is coupled toany one of X₁-X₂₁, X₂₃, X₂₄, X₂₆ to X₂₈, X₃₀ to X₃₂, X₃₄ to X₃₆, and A.77. The method of claim 72, wherein said reactive group is coupled tosaid peptide or derivative by means of a linking group.
 78. The methodof claim 75, wherein X₁ is absent, said reactive group, linkinggroup-(reactive group), tyr or ala, said tyr or ala being optionallycoupled to said reactive group or to said linking group-(reactivegroup); X₂ is absent, pro, said reactive group or said linkinggroup-(reactive group); X₃ is absent, lys or an analog thereof,(reactive group)-lys, (reactive group)-linking group-lys, (reactivegroup)-lys analog, (reactive group)-linking group-lys analog, ile, leu,or ala, wherein said reactive group is coupled to the free amine of lysor lys analog; X₄ is absent, lys or an analog thereof, (reactivegroup)-lys, (reactive group)-linking group-lys, (reactive group)-lysanalog, (reactive group)-linking group-lys analog, or glu, wherein saidreactive group is coupled to the free amine of lys or lys analog; X₅ isabsent or pro; X₆ is absent, glu, val or asp; X₇ is absent, ala, tyr orasn; X₈ is absent or pro; X₉ is absent or gly; X₁₀ is absent, glu orasp; X₁₁ is absent, asp or asn; X₁₂ is absent, lys or an analog thereof,(reactive group)-lys, (reactive group)-linking group-lys, (reactivegroup)-lys analog, (reactive group)-linking group-lys analog, or ala,wherein said reactive group is coupled to the free amine of lys or lysanalog; X₁₃ is absent, lys or an analog thereof, (reactive group)-lys,(reactive group)-linking group-lys, (reactive group)-lys analog,(reactive group)-linking group-lys analog, ser, thr, or pro, whereinsaid reactive group is coupled to the free amine of lys or lys analog;X₁₄ is absent, lys or an analog thereof, (reactive group)-lys, (reactivegroup)-linking group-lys, (reactive group)-lys analog, (reactivegroup)-linking group-lys analog, ala or pro, wherein said reactive groupis coupled to the free amine of lys or lys analog; X₁₅ is absent, lys oran analog thereof, (reactive group)-lys, (reactive group)-linkinggroup-lys, (reactive group)-lys analog, (reactive group)-linkinggroup-lys analog, or glu, wherein said reactive group is coupled to thefree amine of lys or lys analog; X₁₆ is absent, glu, gln or asp; X₁₇ isabsent, leu or met; X₁₈ is absent, lys or an analog thereof, (reactivegroup)-lys, (reactive group)-linking group-lys, (reactive group)-lysanalog, (reactive group)-linking group-lys analog, ser, or ala, whereinsaid reactive group is coupled to the free amine of lys or lys analog;X₁₉ is absent, arg or gln; X₂₀ is absent or tyr; X₂₁ is absent, tyr,ala, a reactive group, or linking group-(reactive group), wherein saidlinking group is coupled to X₂₀ and X₂₂; X₂₂ is ala or ser; X₂₃ is ser,asp or ala; X₂₄ is leu; X₂₅ is arg or lys; X₂₆ is his, arg or lys; X₂₇is tyr; X₂₈ is leu or ile; X₂₉ is asn X₃₀ is leu or met; X₃₁ is val, leuor ile; X₃₂ is thr; X₃₃ is arg or lys; X₃₄ is gln or pro; X₃₅ is arg orlys; X₃₆ is tyr or a derivative thereof; and A is absent, lys or aderivative thereof, (reactive group)-lys, (reactive group)-linkinggroup-lys, (reactive group)-lys derivative, (reactive group)-linkinggroup-lys derivative, where said reactive group is coupled to the freeamine of lys or lys derivative.
 79. The method of claim 78, wherein X₁is absent; X₂ is absent; X₃ leu; X₄ is lys, (reactive group)-lys or(reactive group)-linking group-lys; X₅ is pro; X₆ is glu; X₇ is ala; X₈is pro; X₉ is gly; X₁₀ is glu; X₁₁ is asp; X₁₂ is ala; X₁₃ is ser; X₁₄is pro; X₁₅ is glu; X₁₆ is glu; X₁₇ is leu; X₁₈ is ser; X₁₉ is arg; X₂₀is tyr; X₂₁ is tyr; X₂₂ is ala; X₂₃ is ser; X₂₄ is leu; X₂₅ is arg; X₂₆is his; X₂₇ is tyr; X₂₈ is leu; X₂₉ is asn; X₃₀ is leu; X₃₁ is val; X₃₂is thr; X₃₃ is arg; X₃₄ is gln; X₃₅ is arg; X₃₆ is tyr or a derivativethereof; A is absent, lys or a derivative thereof, (reactive group)-lys,(reactive group)-linking group-lys, (reactive group)-lys derivative,(reactive group)-linking group-lys derivative, where said reactive groupis coupled to the free amine of lys or lys derivative.
 80. The method ofclaim 72, wherein said reactive group is a Michael acceptor, asuccinimidyl-containing group, a maleimido-containing group or anelectrophilic thiol acceptor.
 81. The method of claim 72, wherein saidreactive group is a maleimido-containing group.
 82. The method of claim69, wherein said blood component is a protein.
 83. The method of claim69, wherein said protein is albumin
 84. The method of claim 77, whereinsaid linking group is selected from the group consisting of (2-amino)ethoxy acetic acid (AEA), ethylenediamine (EDA), amino ethoxy ethoxysuccinimic acid (AEES), 2-[2-(2-amino)ethoxy)] ethoxy acetic acid(AEEA), AEEA-AEEA, —NH₂—(CH₂)_(n)-COOH where n is an integer from 1 to20; one or more alkyl chains (C₁-C₁₀) saturated or unsaturated whichoptionally comprises oxygen, nitrogen or sulfur atoms motifs, glycine,3-aminopropionic acid (APA), 8-aminooctanoic acid (OA), 4-aminobenzoicacid (APhA), and combinations thereof.
 85. The method of claim 69,wherein said PYY peptide or functional derivative comprises a peptide ofas defined in any one of SEQ IDS NO: 2 to
 13. 86. The method of claim69, wherein said PYY peptide or functional derivative comprises apeptide of SEQ ID NO:
 4. 87. The method of claim 75, wherein the tyrderivative is of formula:

where R₁ is H, a protecting group (PG), a C₁-C₁₀ branched, linear orcyclic alkyl, a phosphate or a sulfate; R₂ and R₃ are same or differentand selected from the group consisting of H, D and I; R₄ is OH, OPG,OR₅, SH, SPG, SR₅, NH₂, NHPG, N(PG)₂, N(R₅)₂, NR₅PG, or NHR₆, where R₅is a C₁-C₁₀ branched, linear or cyclic alkyl, and R₆ is a solid phasesupport, and wherein the lys derivative is of formula:

where R₄ is as previously defined; and n is an integer having a value of0, 1, 2, 3 or
 4. 88. The method of claim 75, wherein the lys analog isof formula:

where n is an integer having a value of 0, 1, 2, 3 or
 4. 89. In a methodfor treating obesity by administering a PYY peptide or a functionalderivative thereof to a patient, the improvement wherein said PYYpeptide or functional derivative thereof is covalently bonded to a bloodcomponent so as to prevent said PYY peptide or functional derivativethereof from crossing the blood brain barrier, thereby enhancing itsanti-obesity activity.